Recovery of steviol glycosides

ABSTRACT

The present invention relates a process for the recovery of one or more steviol glycosides from a steviol glycoside-containing fermentation broth, which method comprises (a) providing a fermentation broth comprising one or more steviol glycosides and one or more non-steviol glycoside components; (b) separating the liquid phase of the broth from the solid phase of the broth; (c) providing an adsorbent resin; (d) contacting the liquid phase of the broth with the adsorbent resin in order to separate at least a portion of the one or more steviol glycosides from the non-steviol glycoside components, thereby to recover one or more steviol glycosides from the fermentation broth containing one or more steviol glycosides. The invention also relates to a purified steviol glycoside composition prepared using such a process.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/908,811, filed 29 Jan. 2016, which is a 371 National StageApplication of PCT/EP2014/066536, filed 31 Jul. 2014, which claimspriority to U.S. patent application Ser. No. 13/956,144, filed 31 Jul.2013. The disclosures of the priority applications are incorporated intheir entirety herein by reference.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.TXT)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (seeMPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-complianttext file (entitled “Sequence_Listing_2919208-370001_ST25.txt” createdon 8 Feb. 2018, and 1,060,392 bytes in size) is submitted concurrentlywith the instant application, and the entire contents of the SequenceListing are incorporated herein by reference.

BACKGROUND Field of the Invention

The present invention relates to a process for the recovery of one ormore steviol glycosides from a steviol glycoside-containing fermentationbroth. The invention also relates to a composition obtainable by such amethod.

Description of Related Art

The worldwide demand for high potency sweeteners is increasing and, withblending of different artificial sweeteners, becoming a standardpractice. However, the demand for alternatives is expected to increase.The leaves of the perennial herb, Stevia rebaudiana Bert., accumulatequantities of intensely sweet compounds known as steviol glycosides.Whilst the biological function of these compounds is unclear, they havecommercial significance as alternative high potency sweeteners, with theadded advantage that Stevia sweeteners are natural plant products.

These sweet steviol glycosides have functional and sensory propertiesthat appear to be superior to those of many high potency sweeteners. Inaddition, studies suggest that stevioside can reduce blood glucoselevels in Type II diabetics and can reduce blood pressure in mildlyhypertensive patients.

Steviol glycosides accumulate in Stevia leaves where they may comprisefrom 10 to 20% of the leaf dry weight. Stevioside and rebaudioside A areboth heat and pH stable and suitable for use in carbonated beverages andmany other foods. Stevioside is between 110 and 270 times sweeter thansucrose, rebaudioside A between 150 and 320 times sweeter than sucrose.In addition, rebaudioside D is also a high-potency diterpene glycosidesweetener which accumulates in Stevia leaves. It may be about 200 timessweeter than sucrose

Currently, steviol glycosides are extracted from the Stevia plant. InStevia, (−)-kaurenoic acid, an intermediate in gibberellic acid (GA)biosynthesis, is converted into the tetracyclic dipterepene steviol,which then proceeds through a multi-step glucosylation pathway to formthe various steviol glycosides. However, yields may be variable andaffected by agriculture and environmental conditions. Also, Steviacultivation requires substantial land area, a long time prior toharvest, intensive labour and additional costs for the extraction andpurification of the glycosides.

New, more standardized, clean single composition, no after-taste,sources of glycosides are required to meet growing commercial demand forhigh potency, natural sweeteners.

SUMMARY

Steviol glycosides may be produced fermentatively in recombinantmicroorganisms as set out in co-pending International patent applicationno. WO2013/110673 (PCT/EP2013/051262).

The current invention relates to simplification and improvement of theprocess of separating and recovering steviol glycosides from afermentation broth comprising one or more such compounds.

The invention thus provides a process in which fermentatively producedsteviol glycosides may be separated away from the other components ofthe fermentation broth. That is to say, the invention relates to amethod for recovering one or more steviol glycosides from a fermentationbroth comprising one or more such compounds. The invention also relatesto compositions prepared using such a process.

The invention generally relates to recovery of steviol glycosides from afermentation broth using a chromatographic process. Accordingly, theinvention relates to a process for the recovery of one or more steviolglycosides from a steviol glycoside-containing fermentation broth, whichmethod comprises

-   -   (a) providing a fermentation broth comprising one or more        steviol glycosides and one or more non-steviol glycoside        components;    -   (b) separating the liquid phase of the broth from the solid        phase of the broth;    -   (c) providing an adsorbent resin, for example in a packed        column;    -   (d) contacting the liquid phase of the broth with the adsorbent        resin in order to separate at least a portion of the one or more        steviol glycosides from the non-steviol glycoside components,    -   thereby to recover one or more steviol glycosides from the        fermentation broth containing one or more steviol glycosides.

The invention also relates to a process for the recovery of one or moresteviol glycosides from a steviol glycoside-containing fermentationbroth, which method comprises

-   -   (a) providing a steviol glycoside-containing fermentation broth;    -   (b) providing an adsorbent resin, for example in a packed column        in an expanded bed mode;    -   (c) contacting the liquid phase of the broth with the adsorbent        resin in order to separate at least a portion of the one or more        steviol glycosides from the non-steviol glycoside components,    -   thereby to recover one or more steviol glycosides from the        fermentation broth containing one or more steviol glycosides.

The invention also relates to:

a solution comprising one or more steviol glycosides obtainable by aprocess according to the invention; and

a composition which comprises, on a dry solids basis, at least about 95%fermentatively-produced Rebaudioside A, Rebaudioside D or RebaudiosideM.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 sets out a schematic representation of the plasmid pUG7-EcoRV.

FIG. 2 sets out a schematic representation of the method by which theERG20, tHMG1 and BTS1 over-expression cassettes are designed (A) andintegrated (B) into the yeast genome. (C) shows the final situationafter removal of the KANMX marker by the Cre recombinase.

FIG. 3 sets out a schematic representation of the ERG9 knock downconstruct. This consists of a 500 bp long 3′ part of ERG9, 98 bp of theTRP1 promoter, the TRP1 open reading frame and terminator, followed by a400 bp long downstream sequence of ERG9. Due to introduction of a XbaIsite at the end of the ERG9 open reading frame the last amino acidchanges into Ser and the stop codon into Arg. A new stop codon islocated in the TPR1 promoter, resulting in an extension of 18 aminoacids.

FIG. 4 sets out a schematic representation of how UGT2 is integratedinto the genome. A. different fragments used in transformation; B.situation after integration; C. situation after expression of Crerecombinase.

FIG. 5 sets out a schematic representation of how the pathway from GGPPto RebA is integrated into the genome. A. different fragments used intransformation; B. situation after integration.

FIG. 6 sets out the elution pattern of extract (1^(st) run).

FIG. 7 sets out the elution pattern of extract (2^(nd) run).

FIG. 8 sets out a schematic diagram of the potential pathways leading tobiosynthesis of steviol glycosides.

DESCRIPTION OF THE SEQUENCE LISTING

A description of the sequences is set out in Table 1. Sequencesdescribed herein may be defined with reference to the sequence listingor with reference to the database accession numbers also set out inTable 1.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

Throughout the present specification and the accompanying claims, thewords “comprise”, “include” and “having” and variations such as“comprises”, “comprising”, “includes” and “including” are to beinterpreted inclusively. That is, these words are intended to convey thepossible inclusion of other elements or integers not specificallyrecited, where the context allows.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to one or at least one) of the grammatical object of thearticle. By way of example, “an element” may mean one element or morethan one element.

Herein, the term non-steviol glycoside should be taken to mean asubstance which is not a steviol glycoside.

The invention concerns a process for the recovery of one or more steviolglycosides from a steviol glycoside-containing fermentation broth, whichmethod comprises

-   -   (a) providing a fermentation broth comprising one or more        steviol glycosides and one or more non-steviol glycoside        components;    -   (b) separating a liquid phase of the broth from a solid phase of        the broth;    -   (c) contacting the liquid phase of the broth with an adsorbent        resin in order to separate at least a portion of the one or more        steviol glycosides from the non-steviol glycoside components,    -   thereby to recover one or more steviol glycosides from the        fermentation broth containing one or more steviol glycosides.

Typically, the adsorbent resin is provided in a packed column.

The invention also relates to a process for the recovery of one or moresteviol glycosides from a steviol glycoside-containing fermentationbroth, which method comprises

-   -   (a) providing a steviol glycoside-containing fermentation broth;    -   (b) contacting the broth with an adsorbent resin in order to        separate at least a portion of the one or more steviol        glycosides from the non-steviol glycoside components,    -   thereby to recover one or more steviol glycosides from the        fermentation broth containing one or more steviol glycosides.

Typically, the adsorbent resin is provided in a packed column in anexpanded bed mode.

The fermentation broth is a fermentation broth obtained fromfermentation of a microorganism, typically a recombinant microorganism,which is capable of producing one or more steviol glycosides. Suchmicroorganisms and their fermentation are described herein. Typically,the recombinant microorganism is one which is capable of extracellularproduction of one or more steviol glycosides.

Typically, the broth is treated prior to be applied to a chromatographycolumn.

In particular, cells may be disrupted and the resulting solid and liquidphases separated. Cell disruption may be carried out, for example, bymechanical or heat shock. Such cell disruption may not, however, benecessary of the microorganism produced sufficient extracellular steviolglycoside(s). Solid/liquid separation may be carried out, for example,by centrifugation, membrane filtration or microfiltration.

The liquid may then conveniently be applied to a chromatography column.

An alternative separation of liquid and solid phases may comprisespray-drying the broth (for example a broth where the cells have beendisrupted) and then extracting steviol glycosides with a suitablesolvent, for example ethanol. In terms of this invention, this type ofprocess should be understood to constitute “separating a liquid phase ofthe broth from a solid phase of the broth”. The resulting liquid maythen conveniently be applied to a chromatography column.

The process of the invention may alternatively be carried out with wholebroth (i.e. including cells) where the process is carried out in theexpanded bed format. Expanded-bed adsorption allows the capture ofproteins from particle-containing feedstocks without prior removal ofparticulates, thus enabling clarification of a cell suspension or cellhomogenate and the concentration of the desired product in a singleoperation. Another aspect of using the expanded mode is the possibilityof in situ removal of steviol glycosides from the broth whilst cells andnon-bound nutrients are returned back to the fermentation tank.

In the process of the invention, the adsorbent resin may be any suitableresin, for example is a polystyrene-divinylbenzene resin, apolymethacrylate resin, a polyaromatic resin, a functionalizedpolymethacrylate-divinybenzene resin, a functionalizedpolystyrene-divinylbenzene resin or an amino (NH2) bondedmethacrylate/divinylbenzene copolymer resin.

The adsorbant resin may be functionalized with tertiary amines orquaternary amines.

In a process of the invention, the adsorbent may have a surface area ofabout 900 m²/gram or greater.

The process according to the invention may be carried out in anadsorb/desorb chromatography format. In this format, the methodcomprises

-   -   (a) providing a liquid phase (derived from a fermentation broth)        or a fermentation broth and a solvent;    -   (b) providing an adsorbent resin;    -   (c) providing an elution solvent;    -   (d) contacting the adsorbent resin with the liquid phase or        broth and elution solvent so that at least a portion of the        non-steviol glycoside components adsorbs onto the adsorbent        enriching the glycoside solution in steviol glycosides and        resulting in the formation of a purified steviol glycoside        composition that is eluted from the adsorbent along with the        elution solvent; and    -   (e) optionally, desorbing the non-steviol glycoside components        from the adsorbent.

Typically, the adsorbent resin is provided in a packed column.

In such a process, the elution solvent may comprise about 20% weight orless of an alcohol and about 80% weight or greater water.

In such a process, the elution solvent may comprise about 50% weight orless of an alcohol and about 50% weight or greater water.

The process of the invention may be carried out in a format wherein themethod of separating comprises fractionation chromatography. Such aprocess may comprise the steps of:

-   -   (a) providing a liquid phase (derived from a fermentation broth)        or a fermentation broth and a solvent;    -   (b) providing a column packed with an adsorbent; and    -   (c) contacting the adsorbent with the liquid phase or broth so        that at least a portion of the non-steviol glycoside components        adsorb onto the adsorbent and so that at least a portion of the        steviol glycoside adsorbs onto the adsorbent, wherein the        steviol glycosides propagate through the adsorbent at a faster        rate than the non-steviol glycosides; and    -   (d) collecting a steviol glycoside-containing solution from the        adsorbent.

In such a process, the solvent may comprises about 20% weight or greaterof an alcohol and about 80% weight or less water.

In such a process according to claim 9, wherein the solvent comprisesabout 25% to about 35% weight of an alcohol and about 65% to about 75%water.

In such a process, the alcohol may be methanol, ethanol, propanol orbutanol.

In such a process, the solvent may comprise water and the adsorbent maybe a strongly acidic cationic exchange resin.

In any format of the invention, more than one chromatographic cycle maybe carried out, for example two, three, four, five or morechromatographic cycles.

In a process of the invention where two or more chromatographic cyclesare used, chromatography at pH as such may be followed by chromatographyat about pH 8.5 to reduce the concentration of Reb B. Reb B is one ofvery few rebaudiosides that had free carboxy group. Accordingly, at highpH, where this group is charged, RebB will have much lower affinity fora hydrophobic adsorbent (for example HP-20) and hence will not bind toit at pH 8.5 while other non-charged rebaudiosides will still bind well.

The process of the invention permits a purified steviol-glycosidecomprising solution to be recovered. The recovered steviolglycoside-containing solution typically has a purity that is at leastabout 10% greater, at least about 20% greater, at least about about 30%as compared to a purity of the liquid phase or broth (from which the atleast one steviol glycoside is recovered).

Herein, the phrase “separate at least a portion of the one or moresteviol glycosides from the non-steviol glycoside components” should beunderstood to imply that at least a portion of the one or more steviolglycosides is separated from at least a portion of the non-steviolglycoside components. The phrase is not intended to imply that theportion of the one or more steviol glycosides recovered according to theprocess of the invention is necessarily entirely free from non-steviolglycoside components. It is possible that non-steviol glycosidecomponents are recovered too. However, the recovered one or more steviolglycosides should be enriched for the one or more steviol glycosides ascompared with the starting material, eg, a fermentation broth. That isto say, the one or more steviol glycosides recovered according to theinvention should comprise less non-steviol glycosides as compared withthe starting material.

In a process of the invention, the purified steviol glycoside-containingsolution comprises, on a dry solids basis, at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, at least about 95%,at least about 99% weight of Rebaudioside A, Rebaudioside D orRebaudioside M.

The solution may be further processed to a solid form, for example agranulate or power, for example by spray-drying or crystallization. Sucha solid composition may comprise at least about 60%, at least about 70%,at least about 80%, at least about 90%, at least about 95%, at leastabout 99% by weight of Rebaudioside A, Rebaudioside D or Rebaudioside M.

The invention thus provides a solution comprising one or more steviolglycosides obtainable by a process according to the invention. Such asolution may comprises one or more of steviolmonoside, steviolbioside,stevioside or rebaudioside A, rebaudioside B, rebaudioside C,rebaudioside D, rebaudioside E, rebaudioside F, rubusoside, dulcoside Aor rebaudioside M.

Such a solution may comprise, on a dry solids basis, at least about 60%,at least about 70%, at least about 80%, at least about 90%, at leastabout 95%, at least about 99% weight of Rebaudioside A, Rebaudioside Dor Rebaudioside M.

Accordingly, the invention provides a composition which may comprise, ona dry solids basis, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 95%, at least about 99%weight of fermentatively-produced Rebaudioside A, Rebaudioside D orRebaudioside M.

Such a composition may be a granulate or powder obtainable by a processas set out above which includes a step of processing the purifiedsteviol-comprising solution to a solid form. Such a solid compositionmay comprise at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 95%, at least about 99% by weight offermentatively-produced Rebaudioside A, Rebaudioside D or RebaudiosideM.

In the invention, the broth may be derived from the fermentation of anymicroorganism capable of producing a steviol glycoside.

In particular, a broth may be derived from a recombinant microorganismthat is capable of producing a steviol glycoside. Suitable recombinantmicroorganisms are described herein below. Such a recombinantmicroorganism may comprise one or more nucleotide sequence(s) encoding:

a polypeptide having ent-copalyl pyrophosphate synthase activity;

-   -   a polypeptide having ent-Kaurene synthase activity;    -   a polypeptide having ent-Kaurene oxidase activity;    -   a polypeptide having kaurenoic acid 13-hydroxylase activity; and    -   one or more polypeptides having UDP-glucosyltransferase (UGT)        activity,

whereby expression of the nucleotide sequence(s) confer(s) on themicroorganism the ability to produce at least one steviol glycoside.

For the purposes of this invention, a polypeptide having ent-copalylpyrophosphate synthase (EC 5.5.1.13) is capable of catalyzing thechemical reaction:

This enzyme has one substrate, geranylgeranyl pyrophosphate, and oneproduct, ent-copalyl pyrophosphate. This enzyme participates ingibberellin biosynthesis. This enzyme belongs to the family ofisomerase, specifically the class of intramolecular lyases. Thesystematic name of this enzyme class is ent-copalyl-diphosphate lyase(decyclizing). Other names in common use include having ent-copalylpyrophosphate synthase, ent-kaurene synthase A, and ent-kaurenesynthetase A.

For the purposes of this invention, a polypeptide having ent-kaurenesynthase activity (EC 4.2.3.19) is a polypeptide that is capable ofcatalyzing the chemical reaction:

ent-copalyl diphosphate

ent-kaurene+diphosphate

Hence, this enzyme has one substrate, ent-copalyl diphosphate, and twoproducts, ent-kaurene and diphosphate.

This enzyme belongs to the family of lyases, specifically thosecarbon-oxygen lyases acting on phosphates. The systematic name of thisenzyme class is ent-copalyl-diphosphate diphosphate-lyase (cyclizing,ent-kaurene-forming). Other names in common use include ent-kaurenesynthase B, ent-kaurene synthetase B, ent-copalyl-diphosphatediphosphate-lyase, and (cyclizing). This enzyme participates inditerpenoid biosynthesis.

ent-copalyl diphosphate synthases may also have a distinct ent-kaurenesynthase activity associated with the same protein molecule. Thereaction catalyzed by ent-kaurene synthase is the next step in thebiosynthetic pathway to gibberellins. The two types of enzymic activityare distinct, and site-directed mutagenesis to suppress the ent-kaurenesynthase activity of the protein leads to build up of ent-copalylpyrophosphate.

Accordingly, a single nucleotide sequence may encode a polypeptidehaving ent-copalyl pyrophosphate synthase activity and ent-kaurenesynthase activity. Alternatively, the two activities may be encoded twodistinct, separate nucleotide sequences.

For the purposes of this invention, a polypeptide having ent-kaureneoxidase activity (EC 1.14.13.78) is a polypeptide which is capable ofcatalysing three successive oxidations of the 4-methyl group ofent-kaurene to give kaurenoic acid. Such activity typically requires thepresence of a cytochrome P450.

For the purposes of the invention, a polypeptide having kaurenoic acid13-hydroxylase activity (EC 1.14.13) is one which is capable ofcatalyzing the formation of steviol (ent-kaur-16-en-13-ol-19-oic acid)using NADPH and O₂. Such activity may also be referred to as ent-ka13-hydroxylase activity.

A recombinant microorganism which may be fermented to produce afermentation broth for use in the process of the invention comprises oneor more nucleotide sequences encoding a polypeptide havingUDP-glucosyltransferase (UGT) activity, whereby expression of thenucleotide sequence(s) confer(s) on the microorganism the ability toproduce at least one of steviolmonoside, steviolbioside, stevioside orrebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D,rebaudioside E, rebaudioside F, rubusoside, dulcoside A or rebaudiosideM.

For the purposes of this invention, a polypeptide having UGT activity isone which has glycosyltransferase activity (EC 2.4), i.e. that can actas a catalyst for the transfer of a monosaccharide unit from anactivated nucleotide sugar (also known as the “glycosyl donor”) to aglycosyl acceptor molecule, usually an alcohol. The glycosyl donor for aUGT is typically the nucleotide sugar uridine diphosphate glucose(uracil-diphosphate glucose, UDP-glucose).

The UGTs used may be selected so as to produce a desired diterpeneglycoside, such as a steviol glycoside. Schematic diagrams of steviolglycoside formation are set out in Humphrey et al., Plant MolecularBiology (2006) 61: 47-62 and Mohamed et al., J. Plant Physiology 168(2011) 1136-1141. In addition, FIG. 8 sets out a schematic diagram ofsteviol glycoside formation.

The biosynthesis of rebaudioside A involves glucosylation of theaglycone steviol. Specifically, rebaudioside A can be formed byglucosylation of the 13-OH of steviol which forms the13-O-steviolmonoside, glucosylation of the C-2′ of the 13-O-glucose ofsteviolmonoside which forms steviol-1,2-bioside, glucosylation of theC-19 carboxyl of steviol-1,2-bioside which forms stevioside, andglucosylation of the C-3′ of the C-13-O-glucose of stevioside. The orderin which each glucosylation reaction occurs can vary—see FIG. 8. One UGTmay be capable of catalyzing more than one conversion as set out in thisscheme.

Conversion of steviol to rebaudioside A or rebaudioside D may beaccomplished in a recombinant host by the expression of gene(s) encodingthe following functional UGTs: UGT74G1, UGT85C2, UGT76G1 and UGT2. Thus,a recombinant microorganism expressing these four UGTs can makerebaudioside A if it produces steviol or when fed steviol in the medium.Typically, one or more of these genes are recombinant genes that havebeen transformed into a microorganism that does not naturally possessthem. Examples of all of these enzmyes are set out in Table 1. Arecombinant microorganism may comprise any combination of a UGT74G1,UGT85C2, UGT76G1 and UGT2. In Table 1 UGT64G1 sequences are indicated asUGT1 sequences, UGT74G1 sequences are indicated as UGT3 sequences andUGT76G1 sequences are indicated as UGT4 sequences. UGT2 sequences areindicated as UGT2 sequences in Table 1.

A recombinant microorganism which comprises a nucleotide sequenceencoding a polypeptide having UGT activity may comprise a nucleotidesequence encoding a polypeptide capable of catalyzing the addition of aC-13-glucose to steviol. That is to say, a recombinant microorganism maycomprise a UGT which is capable of catalyzing a reaction in whichsteviol is converted to steviolmonoside. Accordingly, expression of sucha nucleotide sequence may confer on the microorganism the ability toproduce at least steviolmonoside.

Such a microorganism may comprise a nucleotide sequence encoding apolypeptide having the activity shown by UDP-glycosyltransferase (UGT)UGT85C2, whereby the nucleotide sequence upon transformation of themicroorganism confers on the cell the ability to convert steviol tosteviolmonoside.

UGT85C2 activity is transfer of a glucose unit to the 13-OH of steviol.Thus, a suitable UGT85C2 may function as a uridine 5′-diphosphoglucosyl: steviol 13-OH transferase, and a uridine 5′-diphosphoglucosyl: steviol-19-0-glucoside 13-OH transferase. A functional UGT85C2polypeptides may also catalyze glucosyl transferase reactions thatutilize steviol glycoside substrates other than steviol andsteviol-19-O-glucoside. Such sequences are indicated as UGT1 sequencesin Table 1.

A recombinant microorganism which comprises a nucleotide sequenceencoding a polypeptide having UGT activity may comprise a nucleotidesequence encoding a polypeptide capable of catalyzing the addition of aC-13-glucose to steviol or steviolmonoside. That is to say, arecombinant microorganism may comprise a UGT which is capable ofcatalyzing a reaction in which steviolmonoside is converted tosteviolbioside. Accordingly, such a microorganism may be capable ofconverting steviolmonoside to steviolbioside. Expression of such anucleotide sequence may confer on the microorganism the ability toproduce at least steviolbioside.

A suitable recombinant microorganism may also comprise a nucleotidesequence encoding a polypeptide having the activity shown byUDP-glycosyltransferase (UGT) UGT74G1, whereby the nucleotide sequenceupon transformation of the microorganism confers on the cell the abilityto convert steviolmonoside to steviolbioside.

A suitable recombinant microorganism may also comprise a nucleotidesequence encoding a polypeptide having the activity shown byUDP-glycosyltransferase (UGT) UGT2, whereby the nucleotide sequence upontransformation of the microorganism confers on the cell the ability toconvert steviolmonoside to steviolbioside.

A suitable UGT2 polypeptide functions as a uridine 5′-diphosphoglucosyl: steviol-13-O-glucoside transferase (also referred to as asteviol-13-monoglucoside 1,2-glucosylase), transferring a glucose moietyto the C-2′ of the 13-O-glucose of the acceptor molecule,steviol-13-O-glucoside. Typically, a suitable UGT2 polypeptide alsofunctions as a uridine 5′-diphospho glucosyl: rubusoside transferasetransferring a glucose moiety to the C-2′ of the 13-O-glucose of theacceptor molecule, rubusoside.

Functional UGT2 polypeptides may also catalyze reactions that utilizesteviol glycoside substrates other than steviol-13-0-glucoside andrubusoside, e.g., functional UGT2 polypeptides may utilize stevioside asa substrate, transferring a glucose moiety to the C-2′ of the19-O-glucose residue to produce Rebaudioside E. A functional UGT2polypeptides may also utilize Rebaudioside A as a substrate,transferring a glucose moiety to the C-2′ of the 19-O-glucose residue toproduce Rebaudioside D. However, a functional UGT2 polypeptide typicallydoes not transfer a glucose moiety to steviol compounds having a1,3-bound glucose at the C-13 position, i.e., transfer of a glucosemoiety to steviol 1,3-bioside and 1,3-stevioside does not occur.

Functional UGT2 polypeptides may also transfer sugar moieties fromdonors other than uridine diphosphate glucose. For example, a functionalUGT2 polypeptide may act as a uridine 5′-diphospho D-xylosyl:steviol-13-O-glucoside transferase, transferring a xylose moiety to theC-2′ of the 13-O-glucose of the acceptor molecule,steviol-13-O-glucoside. As another example, a functional UGT2polypeptide can act as a uridine 5′-diphospho L-rhamnosyl:steviol-13-0-glucoside transferase, transferring a rhamnose moiety tothe C-2′ of the 13-O-glucose of the acceptor molecule,steviol-13-O-glucoside. Such sequences are indicated as UGT2 sequencesin Table 1.

A recombinant microorganism which may be fermented to produce afermentation broth for use in a process of the invention which comprisesa nucleotide sequence encoding a polypeptide having UGT activity maycomprise a nucleotide sequence encoding a polypeptide capable ofcatalyzing the addition of a C-19-glucose to steviolbioside. That is tosay, a suitable recombinant microorganism may comprise a UGT which iscapable of catalyzing a reaction in which steviolbioside is converted tostevioside. Accordingly, such a microorganism may be capable ofconverting steviolbioside to stevioside. Expression of such a nucleotidesequence may confer on the microorganism the ability to produce at leaststevioside.

A suitable recombinant microorganism may also comprise a nucleotidesequence encoding a polypeptide having the activity shown byUDP-glycosyltransferase (UGT) UGT74G1, whereby the nucleotide sequenceupon transformation of the microorganism confers on the cell the abilityto convert steviolbioside to stevioside.

Suitable UGT74G1 polypeptides may be capable of transferring a glucoseunit to the 13-OH or the 19-COOH, respectively, of steviol. A suitableUGT74G1 polypeptide may function as a uridine 5′-diphospho glucosyl:steviol 19-COOH transferase and a uridine 5′-diphospho glucosyl:steviol-13-O-glucoside 19-COOH transferase. Functional UGT74G1polypeptides also may catalyze glycosyl transferase reactions thatutilize steviol glycoside substrates other than steviol andsteviol-13-O-glucoside, or that transfer sugar moieties from donorsother than uridine diphosphate glucose. Such sequences are indicated asUGT1 sequences in Table 3.

A recombinant microorganism which comprises a nucleotide sequenceencoding a polypeptide having UGT activity may comprise a nucleotidesequence encoding a polypeptide capable of catalyzing glucosylation ofthe C-3′ of the glucose at the C-13 position of stevioside. That is tosay, a recombinant microorganism may comprise a UGT which is capable ofcatalyzing a reaction in which stevioside to rebaudioside A.Accordingly, such a microorganism may be capable of convertingstevioside to rebaudioside A. Expression of such a nucleotide sequencemay confer on the microorganism the ability to produce at leastrebaudioside A.

A suitable recombinant microorganism may also comprise a nucleotidesequence encoding a polypeptide having the activity shown byUDP-glycosyltransferase (UGT) UGT76G1, whereby the nucleotide sequenceupon transformation of the microorganism confers on the cell the abilityto convert stevioside to rebaudioside A.

A suitable UGT76G1 adds a glucose moiety to the C-3′ of theC-13-O-glucose of the acceptor molecule, a steviol 1,2 glycoside. Thus,UGT76G1 functions, for example, as a uridine 5′-diphospho glucosyl:steviol 13-0-1,2 glucoside C-3 ‘ glucosyl transferase and a uridine5’-diphospho glucosyl: steviol-19-0-glucose, 13-0-1,2 bioside C-3′glucosyl transferase. Functional UGT76G1 polypeptides may also catalyzeglucosyl transferase reactions that utilize steviol glycoside substratesthat contain sugars other than glucose, e.g., steviol rhamnosides andsteviol xylosides. Such sequences are indicated as UGT4 sequences inTable 1.

A recombinant microorganism may comprise nucleotide sequences encodingpolypeptides having one or more of the four UGT activities describedabove. Preferably, a recombinant microorganism may comprise nucleotidesequences encoding polypeptides having all four of the UGT activitiesdescribed above. A given nucleic acid may encode a polypeptide havingone or more of the above activities. For example, a nucleic acid encodefor a polypeptide which has two, three or four of the activities set outabove. Preferably, a recombinant microorganism comprises UGT1, UGT2 andUGT3 activity. More preferably, such a recombinant microorganism willalso comprise UGT4 activity.

A recombinant microorganism which comprises a nucleotide sequenceencoding a polypeptide having UGT activity may comprise a nucleotidesequence encoding a polypeptide capable of catalyzing the glucosylationof stevioside or rebaudioside A. That is to say, a recombinantmicroorganism may comprise a UGT which is capable of catalyzing areaction in which stevioside or rebaudioside A is converted torebaudioside D. Accordingly, such a microorganism may be capable ofconverting stevioside or rebaudioside A to rebaudioside D. Expression ofsuch a nucleotide sequence may confer on the microorganism the abilityto produce at least rebaudioside D. We have shown that a microorganismexpression a combination of UGT85C2, UGT2, UGT74G1 and UGT76G1polypeptides may be capable of rebaudioside D production.

A microorganism which comprises a nucleotide sequence encoding apolypeptide having UGT activity may comprise a nucleotide sequenceencoding a polypeptide capable of catalyzing the glucosylation ofstevioside. That is to say, a microorganism may comprise a UGT which iscapable of catalyzing a reaction in which stevioside is converted torebaudioside E. Accordingly, such a microorganism may be capable ofconverting stevioside to rebaudioside E. Expression of such a nucleotidesequence may confer on the microorganism the ability to produce at leastrebaudioside E.

A microorganism which comprises a nucleotide sequence encoding apolypeptide having UGT activity may comprise a nucleotide sequenceencoding a polypeptide capable of catalyzing the glucosylation ofrebaudioside E. That is to say, a microorganism may comprise a UGT whichis capable of catalyzing a reaction in which rebaudioside E is convertedto rebaudioside D. Accordingly, such a microorganism may be capable ofconverting stevioside or rebaudioside A to rebaudioside D. Expression ofsuch a nucleotide sequence may confer on the microorganism the abilityto produce at least rebaudioside D.

A recombinant microorganism may be capable of expressing a nucleotidesequence encoding a polypeptide having NADPH-cytochrome p450 reductaseactivity. That is to say, a recombinant microorganism may comprisesequence encoding a polypeptide having NADPH-cytochrome p450 reductaseactivity.

A polypeptide having NADPH-Cytochrome P450 reductase activity (EC1.6.2.4; also known as NADPH:ferrihemoprotein oxidoreductase,NADPH:hemoprotein oxidoreductase, NADPH:P450 oxidoreductase, P450reductase, POR, CPR, CYPOR) is typically one which is a membrane-boundenzyme allowing electron transfer to cytochrome P450 in the microsome ofthe eukaryotic cell from a FAD- and FMN-containing enzymeNADPH:cytochrome P450 reductase (POR; EC 1.6.2.4).

Preferably, a recombinant microorganism, capable of being fermented toprepare a fermentation broth suitable for use in the process of theinvention, is capable of expressing one or more of:

-   -   a. a nucleotide sequence encoding a polypeptide having        NADPH-cytochrome p450 reductase activity, wherein said        nucleotide sequence comprises:        -   i. a nucleotide sequence encoding a polypeptide having            NADPH-cytochrome p450 reductase activity, said polypeptide            comprising an amino acid sequence that has at least about            20%, preferably at least 25, 30, 40, 50, 55, 60, 65, 70, 75,            80, 85, 90, 95, 96, 97, 98, or 99%, sequence identity with            the amino acid sequence of SEQ ID NOs: 54, 56, 58 or 78;        -   ii. a nucleotide sequence that has at least about 15%,            preferably at least 20, 25, 30, 40, 50, 55, 60, 65, 70, 75,            80, 85, 90, 95, 96, 97, 98, or 99%, sequence identity with            the nucleotide sequence of SEQ ID NOs: 53, 55, 57 or 77;        -   iii. a nucleotide sequence the complementary strand of which            hybridizes to a nucleic acid molecule of sequence of (i) or            (ii); or        -   iv. a nucleotide sequence which differs from the sequence of            a nucleic acid molecule of (i), (ii) or (iii) due to the            degeneracy of the genetic code,

Preferably, a recombinant microorganism is one which is capable ofexpressing one or more of:

-   -   a. a nucleotide sequence encoding a polypeptide having        ent-copalyl pyrophosphate synthase activity, wherein said        nucleotide sequence comprises:        -   i. a nucleotide sequence encoding a polypeptide having            ent-copalyl pyrophosphate synthase activity, said            polypeptide comprising an amino acid sequence that has at            least about 20%, preferably at least 25, 30, 40, 50, 55, 60,            65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99%, sequence            identity with the amino acid sequence of SEQ ID NOs: 2, 4,            6, 8, 18, 20, 60 or 62;        -   ii. a nucleotide sequence that has at least about 15%,            preferably at least 20, 25, 30, 40, 50, 55, 60, 65, 70, 75,            80, 85, 90, 95, 96, 97, 98, or 99%, sequence identity with            the nucleotide sequence of SEQ ID NOs: 1, 3, 5, 7, 17, 19,            59 or 61, 141, 142, 151, 152, 153, 154, 159, 160, 182 or            184;        -   iii. a nucleotide sequence the complementary strand of which            hybridizes to a nucleic acid molecule of sequence of (i) or            (ii); or        -   iv. a nucleotide sequence which differs from the sequence of            a nucleic acid molecule of (i), (ii) or (iii) due to the            degeneracy of the genetic code,    -   b. a nucleotide sequence encoding a polypeptide having        ent-Kaurene synthase activity, wherein said nucleotide sequence        comprises:        -   i. a nucleotide sequence encoding a polypeptide having            ent-Kaurene synthase activity, said polypeptide comprising            an amino acid sequence that has at least about 20%,            preferably at least 25, 30, 40, 50, 55, 60, 65, 70, 75, 80,            85, 90, 95, 96, 97, 98, or 99%, sequence identity with the            amino acid sequence of SEQ ID NOs: 10, 12, 14, 16, 18, 20,            64 or 66;        -   ii. a nucleotide sequence that has at least about 15%,            preferably at least 20, 25, 30, 40, 50, 55, 60, 65, 70, 75,            80, 85, 90, 95, 96, 97, 98, or 99%, sequence identity with            the nucleotide sequence of SEQ ID NOs: 9, 11, 13, 15, 17,            19, 63, 65, 143, 144, 155, 156, 157, 158, 159, 160, 183 or            184;        -   iii. a nucleotide sequence the complementary strand of which            hybridizes to a nucleic acid molecule of sequence of (i) or            (ii); or        -   iv. a nucleotide sequence which differs from the sequence of            a nucleic acid molecule of (i), (ii) or (iii) due to the            degeneracy of the genetic code,    -   c. a nucleotide sequence encoding a polypeptide having        ent-Kaurene oxidase activity, wherein said nucleotide sequence        comprises:        -   i. a nucleotide sequence encoding a polypeptide having            ent-Kaurene oxidase activity, said polypeptide comprising an            amino acid sequence that has at least about 20%, preferably            at least 25, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,            96, 97, 98, or 99%, sequence identity with the amino acid            sequence of SEQ ID NOs: 22, 24, 26, 68 or 86;        -   ii. a nucleotide sequence that has at least about 15%,            preferably at least 20, 25, 30, 40, 50, 55, 60, 65, 70, 75,            80, 85, 90, 95, 96, 97, 98, or 99%, sequence identity with            the nucleotide sequence of SEQ ID NOs: 21, 23, 25, 67, 85,            145, 161, 162, 163, 180 or 186;        -   iii. a nucleotide sequence the complementary strand of which            hybridizes to a nucleic acid molecule of sequence of (i) or            (ii); or        -   iv. a nucleotide sequence which differs from the sequence of            a nucleic acid molecule of (i), (ii) or (iii) due to the            degeneracy of the genetic code; or    -   d. a nucleotide sequence encoding a polypeptide having kaurenoic        acid 13-hydroxylase activity, wherein said nucleotide sequence        comprises:        -   i. a nucleotide sequence encoding a polypeptide having            kaurenoic acid 13-hydroxylase activity, said polypeptide            comprising an amino acid sequence that has at least about            20%, preferably at least 25, 30, 40, 50, 55, 60, 65, 70, 75,            80, 85, 90, 95, 96, 97, 98, or 99%, sequence identity with            the amino acid sequence of SEQ ID NOs: 28, 30, 32, 34, 70,            90, 92, 94, 96 or 98;        -   ii. a nucleotide sequence that has at least about 15%,            preferably at least 20, 25, 30, 40, 50, 55, 60, 65, 70, 75,            80, 85, 90, 95, 96, 97, 98, or 99%, sequence identity with            the nucleotide sequence of SEQ ID NOs: 27, 29, 31, 33, 69,            89, 91, 93, 95, 97, 146, 164, 165, 166, 167 or 185;        -   iii. a nucleotide sequence the complementary strand of which            hybridizes to a nucleic acid molecule of sequence of (i) or            (ii); or        -   iv. a nucleotide sequence which differs from the sequence of            a nucleic acid molecule of (i), (ii) or (iii) due to the            degeneracy of the genetic code.

In a recombinant microorganism which is capable of expressing anucleotide sequence encoding a polypeptide capable of catalyzing theaddition of a C-13-glucose to steviol, said nucleotide may comprise:

-   -   i. a nucleotide sequence encoding a polypeptide capable of        catalyzing the addition of a C-13-glucose to steviol, said        polypeptide comprising an amino acid sequence that has at least        about 20%, preferably at least 25, 30, 40, 50, 55, 60, 65, 70,        75, 80, 85, 90, 95, 96, 97, 98, or 99%, sequence identity with        the amino acid sequence of SEQ ID NOs: 36, 38 or 72;    -   ii. a nucleotide sequence that has at least about 15%,        preferably at least 20, 25, 30, 40, 50, 55, 60, 65, 70, 75, 80,        85, 90, 95, 96, 97, 98, or 99%, sequence identity with the        nucleotide sequence of SEQ ID NOs: 35, 37, 71, 147, 168, 169 or        189;    -   iii. a nucleotide sequence the complementary strand of which        hybridizes to a nucleic acid molecule of sequence of (i) or        (ii); or    -   iv. a nucleotide sequence which differs from the sequence of a        nucleic acid molecule of (i), (ii) or (iii) due to the        degeneracy of the genetic code.

In a recombinant microorganism which is capable of expressing anucleotide sequence encoding a polypeptide capable of catalyzing theaddition of a glucose at the C-13 position of steviolmonoside (thistypically indicates glucosylation of the C-2′ of theC-13-glucose/13-O-glucose of steviolmonoside), said nucleotide sequencemay comprise:

-   -   i. a nucleotide sequence encoding a polypeptide capable of        catalyzing the addition of a C-13-glucose to steviol or        steviolmonoside, said polypeptide comprising an amino acid        sequence that has at least about 20%, preferably at least 25,        30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or        99%, sequence identity with the amino acid sequence of SEQ ID        NOs: 88, 100, 102, 104, 106, 108, 110 or 112;    -   ii. a nucleotide sequence that has at least about 15%,        preferably at least 20, 25, 30, 40, 50, 55, 60, 65, 70, 75, 80,        85, 90, 95, 96, 97, 98, or 99%, sequence identity with the        nucleotide sequence of SEQ ID NOs: 87, 99, 101, 103, 105, 107,        109, 111, 181 or 192;    -   iii. a nucleotide sequence the complementary strand of which        hybridizes to a nucleic acid molecule of sequence of (i) or        (ii); or    -   iv. a nucleotide sequence which differs from the sequence of a        nucleic acid molecule of (i), (ii) or (iii) due to the        degeneracy of the genetic code.

In a recombinant microorganism which is capable of expressing anucleotide sequence encoding a polypeptide capable of catalyzing theaddition of a glucose at the C-19 position of steviolbioside, saidnucleotide sequence may comprise:

-   -   i. a nucleotide sequence encoding a polypeptide capable of        catalyzing the addition of a glucose at the C-19 position of        steviolbioside, said polypeptide comprising an amino acid        sequence that has at least about 20% sequence identity with the        amino acid sequence of SEQ ID NOs: 40, 42, 44, 46, 48 or 74;    -   ii. a nucleotide sequence that has at least about 15% sequence        identity with the nucleotide sequence of SEQ ID NOs: 39, 41, 43,        45, 47, 73, 148, 170, 171, 172, 173, 174 or 190;    -   iii. a nucleotide sequence the complementary strand of which        hybridizes to a nucleic acid molecule of sequence of (i) or        (ii); or    -   iv. a nucleotide sequence which differs from the sequence of a        nucleic acid molecule of (i), (ii) or (iii) due to the        degeneracy of the genetic code.

In a recombinant microorganism which expresses a nucleotide sequenceencoding a polypeptide capable of catalyzing glucosylation of the C-3′of the glucose at the C-13 position of stevioside, said nucleotidesequence may comprise:

-   -   i. a nucleotide sequence encoding a polypeptide capable of        catalyzing glucosylation of the C-3′ of the glucose at the C-13        position of stevioside, said polypeptide comprising an amino        acid sequence that has at least about 20%, preferably at least        25, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98,        or 99%, sequence identity with the amino acid sequence of SEQ ID        NOs: 50, 52 or 76;    -   ii. a nucleotide sequence that has at least about 15%,        preferably at least 20, 25, 30, 40, 50, 55, 60, 65, 70, 75, 80,        85, 90, 95, 96, 97, 98, or 99%, sequence identity with the        nucleotide sequence of SEQ ID NOs: 49, 51, 75, 149, 175, 176 or        191;    -   iii. a nucleotide sequence the complementary strand of which        hybridizes to a nucleic acid molecule of sequence of (i) or        (ii); or    -   iv. a nucleotide sequence which differs from the sequence of a        nucleic acid molecule of (i), (ii) or (iii) due to the        degeneracy of the genetic code.

In a recombinant microorganism which expresses a nucleotide sequenceencoding a polypeptide capable of catalysing one or more of: theglucosylation of stevioside or rebaudioside A to rebaudioside D; theglucosylation of stevioside to rebaudioside E; or the glucosylation ofrebaudioside E to rebaudioside D, said nucleotide sequence may comprise:

-   -   i. a nucleotide sequence encoding a polypeptide capable of        catalysing one or more of: the glucosylation of stevioside or        rebaudioside A to rebaudioside D; the glucosylation of        stevioside to rebaudioside E; or the glucosylation of        rebaudioside E to rebaudioside D, said polypeptide comprising an        amino acid sequence that has at least about 20% sequence        identity with the amino acid sequence of SEQ ID NOs: 88, 100,        102, 104, 106, 108, 110, 112;    -   ii. a nucleotide sequence that has at least about 15% sequence        identity with the nucleotide sequence of SEQ ID NOs: 87, 99,        101, 103, 105, 107, 109, 111, 181 or 192;    -   iii. a nucleotide sequence the complementary strand of which        hybridizes to a nucleic acid molecule of sequence of (i) or        (ii); or    -   iv. a nucleotide sequence which differs from the sequence of a        nucleic acid molecule of (i), (ii) or (iii) due to the        degeneracy of the genetic code.

A suitable microorganism may be one in which the ability of themicroorganism to produce geranylgeranyl pyrophosphate (GGPP) isupregulated. Upregulated in the context of this invention implies thatthe microorganism produces more GGPP than an equivalent non-transformedstrain.

Accordingly, a suitable recombinant microorganism may comprise one ormore nucleotide sequence(s) encoding hydroxymethylglutaryl-CoAreductase, farnesyl-pyrophosphate synthetase and geranylgeranyldiphosphate synthase, whereby the nucleotide sequence(s) upontransformation of the microorganism confer(s) on the microorganism theability to produce elevated levels of GGPP.

Preferably, a suitable recombinant microorganism is one which is capableof expressing one or more of:

-   -   a. a nucleotide sequence encoding a polypeptide having        hydroxymethylglutaryl-CoA reductase activity, wherein said        nucleotide sequence comprises:        -   i. a nucleotide sequence encoding a polypeptide having            hydroxymethylglutaryl-CoA reductase activity, said            polypeptide comprising an amino acid sequence that has at            least about 20% sequence identity with the amino acid            sequence of SEQ ID NO: 80;        -   ii. a nucleotide sequence that has at least about 15%            sequence identity with the nucleotide sequence of SEQ ID NO:            79;        -   iii. a nucleotide sequence the complementary strand of which            hybridizes to a nucleic acid molecule of sequence of (i) or            (ii); or        -   iv. a nucleotide sequence which differs from the sequence of            a nucleic acid molecule of (i), (ii) or (iii) due to the            degeneracy of the genetic code,    -   b. a nucleotide sequence encoding a polypeptide having        farnesyl-pyrophosphate synthetase activity, wherein said        nucleotide sequence comprises:        -   i. a nucleotide sequence encoding a polypeptide having            farnesyl-pyrophosphate synthetase activity, said polypeptide            comprising an amino acid sequence that has at least about            20% sequence identity with the amino acid sequence of SEQ ID            NO: 82;        -   ii. a nucleotide sequence that has at least about 15%            sequence identity with the nucleotide sequence of SEQ ID            NOs: 81;        -   iii. a nucleotide sequence the complementary strand of which            hybridizes to a nucleic acid molecule of sequence of (i) or            (ii); or        -   iv. a nucleotide sequence which differs from the sequence of            a nucleic acid molecule of (iii) due to the degeneracy of            the genetic code; or c. a nucleotide sequence encoding a            polypeptide having geranylgeranyl diphosphate synthase            activity, wherein said nucleotide sequence comprises:    -   i. a nucleotide sequence encoding a polypeptide having        geranylgeranyl diphosphate synthase activity, said polypeptide        comprising an amino acid sequence that has at least about 20%        sequence identity with the amino acid sequence of SEQ ID NO: 84;    -   ii. a nucleotide sequence that has at least about 15% sequence        identity with the nucleotide sequence of SEQ ID NOs: 83;    -   iii. a nucleotide sequence the complementary strand of which        hybridizes to a nucleic acid molecule of sequence of (i) or        (ii); or    -   iv. a nucleotide sequence which differs from the sequence of a        nucleic acid molecule of (i), (ii) or (iii) due to the        degeneracy of the genetic code.

A microorganism or microbe, for the purposes of this invention, istypically an organism that is not visible to the human eye (i.e.microscopic). A microorganism may be from bacteria, fungi, archaea orprotists. Typically a microorganism will be a single-celled orunicellular organism.

As used herein a recombinant microorganism is defined as a microorganismwhich is genetically modified or transformed/transfected with one ormore of the nucleotide sequences as defined herein. The presence of theone or more such nucleotide sequences alters the ability of themicroorganism to produce a diterpene or diterpene glycoside, inparticular steviol or steviol glycoside. A microorganism that is nottransformed/transfected or genetically modified, is not a recombinantmicroorganism and does typically not comprise one or more of thenucleotide sequences enabling the cell to produce a diterpene orditerpene glycoside. Hence, a non-transformed/non-transfectedmicroorganism is typically a microorganism that does not naturallyproduce a diterpene, although a microorganism which naturally produces aditerpene or diterpene glycoside and which has been modified, asdescribed herein for example (and which thus has an altered ability toproduce a diterpene/diterpene gylcoside), is considered a recombinantmicroorganism.

Sequence identity is herein defined as a relationship between two ormore amino acid (polypeptide or protein) sequences or two or morenucleic acid (polynucleotide) sequences, as determined by comparing thesequences. Usually, sequence identities or similarities are comparedover the whole length of the sequences compared. In the art, “identity”also means the degree of sequence relatedness between amino acid ornucleic acid sequences, as the case may be, as determined by the matchbetween strings of such sequences. “Identity” and “similarity” can bereadily calculated by various methods, known to those skilled in theart. Preferred methods to determine identity are designed to give thelargest match between the sequences tested. Typically then, identitiesand similarities are calculated over the entire length of the sequencesbeing compared. Methods to determine identity and similarity arecodified in publicly available computer programs. Preferred computerprogram methods to determine identity and similarity between twosequences include e.g. the BestFit, BLASTP, BLASTN, and FASTA (Altschul,S. F. et al., J. Mol. Biol. 215:403-410 (1990), publicly available fromNCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIHBethesda, Md. 20894). Preferred parameters for amino acid sequencescomparison using BLASTP are gap open 10.0, gap extend 0.5, Blosum 62matrix. Preferred parameters for nucleic acid sequences comparison usingBLASTP are gap open 10.0, gap extend 0.5, DNA full matrix (DNA identitymatrix).

Nucleotide sequences encoding the enzymes expressed in the cellsdescribed herein may also be defined by their capability to hybridizewith the nucleotide sequences of SEQ ID NO.'s 1, 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81 or 84 it anyother sequence mentioned herein respectively, under moderate, orpreferably under stringent hybridisation conditions. Stringenthybridisation conditions are herein defined as conditions that allow anucleic acid sequence of at least about 25, preferably about 50nucleotides, 75 or 100 and most preferably of about 200 or morenucleotides, to hybridise at a temperature of about 65° C. in a solutioncomprising about 1 M salt, preferably 6×SSC or any other solution havinga comparable ionic strength, and washing at 65° C. in a solutioncomprising about 0.1 M salt, or less, preferably 0.2×SSC or any othersolution having a comparable ionic strength. Preferably, thehybridisation is performed overnight, i.e. at least for 10 hours andpreferably washing is performed for at least one hour with at least twochanges of the washing solution. These conditions will usually allow thespecific hybridisation of sequences having about 90% or more sequenceidentity.

Moderate conditions are herein defined as conditions that allow anucleic acid sequences of at least 50 nucleotides, preferably of about200 or more nucleotides, to hybridise at a temperature of about 45° C.in a solution comprising about 1 M salt, preferably 6×SSC or any othersolution having a comparable ionic strength, and washing at roomtemperature in a solution comprising about 1 M salt, preferably 6×SSC orany other solution having a comparable ionic strength. Preferably, thehybridisation is performed overnight, i.e. at least for 10 hours, andpreferably washing is performed for at least one hour with at least twochanges of the washing solution. These conditions will usually allow thespecific hybridisation of sequences having up to 50% sequence identity.The person skilled in the art will be able to modify these hybridisationconditions in order to specifically identify sequences varying inidentity between 50% and 90%.

The nucleotide sequences encoding an ent-copalyl pyrophosphate synthase;ent-Kaurene synthase; ent-Kaurene oxidase; kaurenoic acid13-hydroxylase; UGT; hydroxymethylglutaryl-CoA reductase,farnesyl-pyrophosphate synthetase; geranylgeranyl diphosphate synthase;NADPH-cytochrome p450 reductase, may be from prokaryotic or eukaryoticorigin.

A nucleotide sequence encoding an ent-copalyl pyrophosphate synthase mayfor instance comprise a sequence as set out in SEQ ID. NO: 1, 3, 5, 7,17, 19, 59, 61, 141, 142, 151, 152, 153, 154, 159, 160, 182 or 184.

A nucleotide sequence encoding an ent-Kaurene synthase may for instancecomprise a sequence as set out in SEQ ID. NO: 9, 11, 13, 15, 17, 19, 63,65, 143, 144, 155, 156, 157, 158, 159, 160, 183 or 184.

A nucleotide sequence encoding an ent-Kaurene oxidase may for instancecomprise a sequence as set out in SEQ ID. NO: 21, 23, 25, 67, 85, 145,161, 162, 163, 180 or 186. A preferred KO is the polypeptide encoded bythe nucleic acid set out in SEQ ID NO: 85.

A nucleotide sequence encoding a kaurenoic acid 13-hydroxylase may forinstance comprise a sequence as set out in SEQ ID. NO: 27, 29, 31, 33,69, 89, 91, 93, 95, 97, 146, 164, 165, 166, 167 or 185. A preferred KAHsequence is the polypeptide encoded by the nucleic acid set out in SEQID NO: 33.

A suitable recombinant microorganism may express a combination of thepolypeptides encoded by SEQ ID NO: 85 and SEQ ID NO: 33 or a variant ofeither thereof as herein described. A preferred recombinantmicroorganism may express the combination of sequences set out in Table8 (in combination with any UGT2, but in particular that encoded by SEQID NO: 87).

A nucleotide sequence encoding a UGT may for instance comprise asequence as set out in SEQ ID. NO: 35, 37, 39, 41, 43, 45, 47, 49, 51,71, 73, 75, 168, 169, 170, 171, 172, 173, 174, 175, 176, 147, 148, 149,87, 181, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,140, 189, 190, 191 or 192.

A nucleotide sequence encoding a hydroxymethylglutaryl-CoA reductase mayfor instance comprise a sequence as set out in SEQ ID. NO: 79.

A nucleotide sequence encoding a farnesyl-pyrophosphate synthetase mayfor instance comprise a sequence as set out in SEQ ID. NO: 81.

A nucleotide sequence encoding a geranylgeranyl diphosphate synthase mayfor instance comprise a sequence as set out in SEQ ID. NO:83.

A nucleotide sequence encoding a NADPH-cytochrome p450 reductase may forinstance comprise a sequence as set out in SEQ ID. NO: 53, 55, 57 or 77.

In the case of the UGT sequences, combinations of at least one from eachof: (i) SEQ ID NOs: 35, 37, 168, 169, 71, 147 or 189; (ii) SEQ ID NOs:87, 99, 101, 103, 105, 107, 109, 111, 181 or 192; (iii) SEQ ID NOs: 39,41, 43, 45, 47, 170, 171, 172, 173, 174, 73, 148 or 190; and (iv) SEQ IDNOs: 49, 51, 175, 176, 75, 149 or 191 may be preferred. Typically, atleast one UGT from group (i) may be used. If at least one UGT from group(iii) is used, generally at least one UGT from group (i) is also used.If at least one UGT from group (iv) is used, generally at least one UGTfrom group (i) and at least one UGT from group (iii) is used. Typically,at least one UGT form group (ii) is used.

A sequence which has at least about 10%, about 15%, about 20%,preferably at least about 25%, about 30%, about 40%, about 50%, about55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,about 90%, about 95%, about 96%, about 97%, about 98%, or about 99%sequence identity with a sequence as mentioned may be used in theinvention.

To increase the likelihood that the introduced enzymes are expressed inactive form in a recombinant microorganism, the corresponding encodingnucleotide sequence may be adapted to optimise its codon usage to thatof the chosen eukaryote host cell. The adaptiveness of the nucleotidesequences encoding the enzymes to the codon usage of the chosen hostcell may be expressed as codon adaptation index (CAI). The codonadaptation index is herein defined as a measurement of the relativeadaptiveness of the codon usage of a gene towards the codon usage ofhighly expressed genes. The relative adaptiveness (w) of each codon isthe ratio of the usage of each codon, to that of the most abundant codonfor the same amino acid. The CAI index is defined as the geometric meanof these relative adaptiveness values. Non-synonymous codons andtermination codons (dependent on genetic code) are excluded. CAI valuesrange from 0 to 1, with higher values indicating a higher proportion ofthe most abundant codons (see Sharp and Li, 1987, Nucleic Acids Research15: 1281-1295; also see: Jansen et al., 2003, Nucleic Acids Res.31(8):2242-51). An adapted nucleotide sequence preferably has a CAI ofat least 0.2, 0.3, 0.4, 0.5, 0.6 or 0.7.

In a preferred embodiment the recombinant is genetically modified with(a) nucleotide sequence(s) which is (are) adapted to the codon usage ofthe eukaryotic cell using codon pair optimisation technology asdisclosed in PCT/EP2007/05594. Codon-pair optimisation is a method forproducing a polypeptide in a host cell, wherein the nucleotide sequencesencoding the polypeptide have been modified with respect to theircodon-usage, in particular the codon-pairs that are used, to obtainimproved expression of the nucleotide sequence encoding the polypeptideand/or improved production of the polypeptide. Codon pairs are definedas a set of two subsequent triplets (codons) in a coding sequence.

Further improvement of the activity of the enzymes in vivo in arecombinant microorganism, can be obtained by well-known methods likeerror prone PCR or directed evolution. A preferred method of directedevolution is described in WO03010183 and WO03010311.

A suitable recombinant microorganism may be any suitable host cell frommicrobial origin. Preferably, the host cell is a yeast or a filamentousfungus. More preferably, the host cell belongs to one of the generaSaccharomyces, Aspergillus, Penicillium, Pichia, Kluyveromyces,Yarrowia, Candida, Hansenula, Humicola, Torulaspora, Trichosporon,Brettanomyces, Pachysolen or Yamadazyma or Zygosaccharomyces.

A more preferred microorganism belongs to the species Aspergillus niger,Penicillium chrysogenum, Pichia stipidis, Kluyveromyces marxianus, K.lactis, K. thermotolerans, Yarrowia lipolytica, Candida sonorensis, C.glabrata, Hansenula polymorpha, Torulaspora delbrueckii, Brettanomycesbruxellensis, Zygosaccharomyces bailii, Saccharomyces uvarum,Saccharomyces bayanus or Saccharomyces cerevisiae species. Preferably,the eukaryotic cell is a Saccharomyces cerevisiae.

A recombinant yeast cell may be modified so that the ERG9 gene isdown-regulated and or the ERG5/ERG6 genes are deleted. Correspondinggenes may be modified in this way in other microorganisms.

Such a microorganism may be transformed, whereby the nucleotidesequence(s) with which the microorganism is transformed confer(s) on thecell the ability to produce a diterpene or glycoside thereof.

A preferred suitable recombinant microorganism is a yeast, such as aSaccharomyces cerevisiae or Yarrowia lipolytica cell. A recombinantmicroorganism, such as a recombinant Saccharomyces cerevisiae cell orYarrowia lipolytica cell may comprise one or more nucleotide sequence(s)from each of the following groups;

(i) SEQ ID. NO: 1, 3, 5, 7, 17, 19, 59, 61, 141, 142, 152, 153, 154,159, 160, 182 or 184.

(ii) SEQ ID. NO: 9, 11, 13, 15, 17, 19, 63, 65, 143, 144, 155, 156, 157,158, 159, 160, 183 or 184.

(iii) SEQ ID. NO: 21, 23, 25, 67 85, 145, 161, 162, 163, 180 or 186.

(iv) SEQ ID. NO: 27, 29, 31, 33, 69, 89, 91, 93, 95, 97, 146, 164, 165,166, 167 or 185.

Such a microorganism will typically also comprise one or more nucleotidesequence(s) as set out in SEQ ID. NO: 53, 55, 57 or 77.

Such a microorganism may also comprise one or more nucleotide sequencesas set out in 35, 37, 39, 41, 43, 45, 47, 49, 51, 71, 73, 75, 168, 169,170, 171, 172, 173, 174, 175, 176, 147, 148, 149, 87, 181, 99, 100, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 189, 190, 191 or192. In the case of these sequences, combinations of at least one fromeach of (i) SEQ ID NOs: 35, 37, 168, 169, 71, 147 or 189; (ii) SEQ IDNOs: 87, 99, 101, 103, 105, 107, 109, 111, 181 or 192; (iii) SEQ ID NOs:39, 41, 43, 45, 47, 170, 171, 172, 173, 174, 73, 148 or 190; and (iv)SEQ ID NOs: 49, 51, 175, 176, 75, 149 or 191 may be preferred.Typically, at least one UGT from group (i) may be used. If at least oneUGT from group (iii) is used, generally at least one UGT from group (i)is also used. If at least one UGT from group (iv) is used, generally atleast one UGT from group (i) and at least one UGT from group (iii) isused. Typically, at least one UGT form group (ii) is used.

Such a microorganism may also comprise the following nucleotidesequences: SEQ ID. NO: 79; SEQ ID. NO: 81; and SEQ ID. NO: 83.

For each sequence set out above (or any sequence mentioned herein), avariant having at least about 15%, preferably at least about 20, about25, about 30, about 40, about 50, about 55, about 60, about 65, about70, about 75, about 80, about 85, about 90, about 95, about 96, about97, about 98, or about 99%, sequence identity with the stated sequencemay be used.

The nucleotide sequences encoding the ent-copalyl pyrophosphatesynthase, ent-Kaurene synthase, ent-Kaurene oxidase, kaurenoic acid13-hydroxylase, UGTs, hydroxymethylglutaryl-CoA reductase,farnesyl-pyrophosphate synthetase, geranylgeranyl diphosphate synthaseand NADPH-cytochrome p450 reductase may be ligated into one or morenucleic acid constructs to facilitate the transformation of themicroorganism.

A nucleic acid construct may be a plasmid carrying the genes encodingenzymes of the diterpene, eg. steviol/steviol glycoside, pathway asdescribed above, or a nucleic acid construct may comprise two or threeplasmids carrying each three or two genes, respectively, encoding theenzymes of the diterpene pathway distributed in any appropriate way.

Any suitable plasmid may be used, for instance a low copy plasmid or ahigh copy plasmid.

It may be possible that the enzymes selected from the group consistingof ent-copalyl pyrophosphate synthase, ent-Kaurene synthase, ent-Kaureneoxidase, and kaurenoic acid 13-hydroxylase, UGTs,hydroxymethylglutaryl-CoA reductase, farnesyl-pyrophosphate synthetase,geranylgeranyl diphosphate synthase and NADPH-cytochrome p450 reductaseare native to the host microorganism and that transformation with one ormore of the nucleotide sequences encoding these enzymes may not berequired to confer the host cell the ability to produce a diterpene orditerpene glycosidase. Further improvement of diterpene/diterpeneglycosidase production by the host microorganism may be obtained byclassical strain improvement.

The nucleic acid construct may be maintained episomally and thuscomprise a sequence for autonomous replication, such as an autosomalreplication sequence sequence. If the host cell is of fungal origin, asuitable episomal nucleic acid construct may e.g. be based on the yeast2μ or pKD1 plasmids (Gleer et al., 1991, Biotechnology 9: 968-975), orthe AMA plasmids (Fierro et al., 1995, Curr Genet. 29:482-489).

Alternatively, each nucleic acid construct may be integrated in one ormore copies into the genome of the host cell. Integration into the hostcell's genome may occur at random by non-homologous recombination butpreferably the nucleic acid construct may be integrated into the hostcell's genome by homologous recombination as is well known in the art(see e.g. WO90/14423, EP-A-0481008, EP-A-0635 574 and U.S. Pat. No.6,265,186).

Optionally, a selectable marker may be present in the nucleic acidconstruct. As used herein, the term “marker” refers to a gene encoding atrait or a phenotype which permits the selection of, or the screeningfor, a microorganism containing the marker. The marker gene may be anantibiotic resistance gene whereby the appropriate antibiotic can beused to select for transformed cells from among cells that are nottransformed. Alternatively or also, non-antibiotic resistance markersare used, such as auxotrophic markers (URA3, TRP1, LEU2). The host cellstransformed with the nucleic acid constructs may be marker gene free.Methods for constructing recombinant marker gene free microbial hostcells are disclosed in EP-A-0 635 574 and are based on the use ofbidirectional markers. Alternatively, a screenable marker such as GreenFluorescent Protein, lacZ, luciferase, chloramphenicolacetyltransferase, beta-glucuronidase may be incorporated into thenucleic acid constructs allowing for screening for transformed cells. Apreferred marker-free method for the introduction of heterologouspolynucleotides is described in WO0540186.

In a preferred embodiment, the nucleotide sequences encoding ent-copalylpyrophosphate synthase, ent-Kaurene synthase, ent-Kaurene oxidase, andkaurenoic acid 13-hydroxylase, UGTs, hydroxymethylglutaryl-CoAreductase, farnesyl-pyrophosphate synthetase, geranylgeranyl diphosphatesynthase and NADPH-cytochrome p450 reductase, are each operably linkedto a promoter that causes sufficient expression of the correspondingnucleotide sequences in the recombinant microorganism to confer to thecell the ability to produce a diterpene or diterpene glycoside.

As used herein, the term “operably linked” refers to a linkage ofpolynucleotide elements (or coding sequences or nucleic acid sequence)in a functional relationship. A nucleic acid sequence is “operablylinked” when it is placed into a functional relationship with anothernucleic acid sequence. For instance, a promoter or enhancer is operablylinked to a coding sequence if it affects the transcription of thecoding sequence.

As used herein, the term “promoter” refers to a nucleic acid fragmentthat functions to control the transcription of one or more genes,located upstream with respect to the direction of transcription of thetranscription initiation site of the gene, and is structurallyidentified by the presence of a binding site for DNA-dependent RNApolymerase, transcription initiation sites and any other DNA sequences,including, but not limited to transcription factor binding sites,repressor and activator protein binding sites, and any other sequencesof nucleotides known to one of skilled in the art to act directly orindirectly to regulate the amount of transcription from the promoter. A“constitutive” promoter is a promoter that is active under mostenvironmental and developmental conditions. An “inducible” promoter is apromoter that is active under environmental or developmental regulation.

The promoter that could be used to achieve the expression of thenucleotide sequences coding for an enzyme as defined herein above, maybe not native to the nucleotide sequence coding for the enzyme to beexpressed, i.e. a promoter that is heterologous to the nucleotidesequence (coding sequence) to which it is operably linked. Preferably,the promoter is homologous, i.e. endogenous to the host cell

Suitable promoters for use in recombinant microorganisms may be GAL7,GAL10, or GAL 1, CYC1, HIS3, ADH1, PGL, PH05, GAPDH, ADC1, TRP1, URA3,LEU2, ENO, TPI, and AOX1. Other suitable promoters include PDC, GPD1,PGK1, TEF1, and TDH.

Any terminator, which is functional in the cell, may be used. Preferredterminators are obtained from natural genes of the host cell. Suitableterminator sequences are well known in the art. Preferably, suchterminators are combined with mutations that prevent nonsense mediatedmRNA decay in the host cell (see for example: Shirley et al., 2002,Genetics 161:1465-1482).

Nucleotide sequences used may include sequences which target them todesired compartments of the microorganism. For example, in a preferredrecombinant microorganism, all nucleotide sequences, except forent-Kaurene oxidase, kaurenoic acid 13-hydroxylase and NADPH-cytochromep450 reductase encoding sequences may be targeted to the cytosol. Thisapproach may be used in a yeast cell.

The term “homologous” when used to indicate the relation between a given(recombinant) nucleic acid or polypeptide molecule and a given hostorganism or host cell, is understood to mean that in nature the nucleicacid or polypeptide molecule is produced by a host cell or organisms ofthe same species, preferably of the same variety or strain.

The term “heterologous” when used with respect to a nucleic acid (DNA orRNA) or protein refers to a nucleic acid or protein that does not occurnaturally as part of the organism, cell, genome or DNA or RNA sequencein which it is present, or that is found in a cell or location orlocations in the genome or DNA or RNA sequence that differ from that inwhich it is found in nature. Heterologous nucleic acids or proteins arenot endogenous to the cell into which it is introduced, but have beenobtained from another cell or synthetically or recombinantly produced.

Typically, a suitable recombinant microorganism will compriseheterologous nucleotide sequences. Alternatively, a recombinantmicroorganism may comprise entirely homologous sequence which has beenmodified as set out herein so that the microorganism produces increasedamounts of a diterpene and/or diterpene glycoside in comparison to anon-modified version of the same microorganism.

One or more enzymes of the diterpene pathway as described herein may beoverexpressed to achieve a sufficient diterpene production by the cell.

There are various means available in the art for overexpression ofenzymes in the host cell. In particular, an enzyme may be overexpressedby increasing the copy number of the gene coding for the enzyme in thehost cell, e.g. by integrating additional copies of the gene in the hostcell's genome.

A preferred recombinant microorganism may be a recombinant microorganismwhich is naturally capable of producing GGPP.

A suitable recombinant microorganism may be able to grow on any suitablecarbon source known in the art and convert it to one or more steviolglycosides. The recombinant microorganism may be able to convertdirectly plant biomass, celluloses, hemicelluloses, pectines, rhamnose,galactose, fucose, maltose, maltodextrines, ribose, ribulose, or starch,starch derivatives, sucrose, lactose and glycerol. Hence, a preferredhost organism expresses enzymes such as cellulases (endocellulases andexocellulases) and hemicellulases (e.g. endo- and exo-xylanases,arabinases) necessary for the conversion of cellulose into glucosemonomers and hemicellulose into xylose and arabinose monomers,pectinases able to convert pectines into glucuronic acid andgalacturonic acid or amylases to convert starch into glucose monomers.Preferably, the host cell is able to convert a carbon source selectedfrom the group consisting of glucose, xylose, arabinose, sucrose,lactose and glycerol. The host cell may for instance be a eukaryotichost cell as described in WO03/062430, WO06/009434, EP1499708B1,WO2006096130 or WO04/099381.

A recombinant microorganism as described above may be used in a processfor the production of a steviol glycoside, which method comprisesfermenting a transformed a suitable recombinant microorganism (asdescribed herein) in a suitable fermentation medium, and optionallyrecovering the diterpene and/or diterpene glycoside.

The fermentation medium used in the process for the production of aditerpene or diterpene glycoside may be any suitable fermentation mediumwhich allows growth of a particular eukaryotic host cell. The essentialelements of the fermentation medium are known to the person skilled inthe art and may be adapted to the host cell selected.

Preferably, the fermentation medium comprises a carbon source selectedfrom the group consisting of plant biomass, celluloses, hemicelluloses,pectines, rhamnose, galactose, fucose, fructose, maltose,maltodextrines, ribose, ribulose, or starch, starch derivatives,sucrose, lactose, fatty acids, triglycerides and glycerol. Preferably,the fermentation medium also comprises a nitrogen source such as ureum,or an ammonium salt such as ammonium sulphate, ammonium chloride,ammoniumnitrate or ammonium phosphate.

A suitable fermentation process may be carried out in batch, fed-batchor continuous mode. A separate hydrolysis and fermentation (SHF) processor a simultaneous saccharification and fermentation (SSF) process mayalso be applied. A combination of these fermentation process modes mayalso be possible for optimal productivity. A SSF process may beparticularly attractive if starch, cellulose, hemicelluose or pectin isused as a carbon source in the fermentation process, where it may benecessary to add hydrolytic enzymes, such as cellulases, hemicellulasesor pectinases to hydrolyse the substrate.

The recombinant microorganism used in the process for the preparation ofa steviol glycoside may be any suitable microorganism as defined hereinabove. It may be advantageous to use a recombinant eukaryoticmicroorganism as described herein in the process for the production of aditerpene or diterpene glycoside, because most eukaryotic cells do notrequire sterile conditions for propagation and are insensitive tobacteriophage infections. In addition, eukaryotic host cells may begrown at low pH to prevent bacterial contamination.

The recombinant microorganism may be a facultative anaerobicmicroorganism. A facultative anaerobic microorganism can be propagatedaerobically to a high cell concentration. This anaerobic phase can thenbe carried out at high cell density which reduces the fermentationvolume required substantially, and may minimize the risk ofcontamination with aerobic microorganisms.

The fermentation process for the production of a steviol glycoside maybe an aerobic or an anaerobic fermentation process.

An anaerobic fermentation process may be herein defined as afermentation process run in the absence of oxygen or in whichsubstantially no oxygen is consumed, preferably less than 5, 2.5 or 1mmol/L/h, and wherein organic molecules serve as both electron donor andelectron acceptors. The fermentation process may also first be run underaerobic conditions and subsequently under anaerobic conditions.

The fermentation process may also be run under oxygen-limited, ormicro-aerobical, conditions. Alternatively, the fermentation process mayfirst be run under aerobic conditions and subsequently underoxygen-limited conditions. An oxygen-limited fermentation process is aprocess in which the oxygen consumption is limited by the oxygentransfer from the gas to the liquid. The degree of oxygen limitation isdetermined by the amount and composition of the ingoing gas flow as wellas the actual mixing/mass transfer properties of the fermentationequipment used.

The production of a steviol glycoside in the fermentation process mayoccur during the growth phase of the host cell, during the stationary(steady state) phase or during both phases. It may be possible to runthe fermentation process at different temperatures.

The process for the production of a steviol glycoside may be run at atemperature which is optimal for the recombinant microorganism. Theoptimum growth temperature may differ for each transformed cell and isknown to the person skilled in the art. The optimum temperature might behigher than optimal for wild type organisms to grow the organismefficiently under non-sterile conditions under minimal infectionsensitivity and lowest cooling cost. Alternatively, the process may becarried out at a temperature which is not optimal for growth of therecombinant microorganism.

The temperature for growth of the recombinant microorganism in a processfor production of a diterpene or diterpene glycoside may be above 20°C., 22° C., 25° C., 28° C., or above 30° C., 35° C., or above 37° C.,40° C., 42° C., and preferably below 45° C. During the production phaseof a diterpene or diterpene glycoside however, the optimum temperaturemight be lower than average in order to optimize biomass stability. Thetemperature during this phase may be below 45° C., for instance below42° C., 40° C., 37° C., for instance below 35° C., 30° C., or below 28°C., 25° C., 22° C. or below 20° C. preferably above 15° C.

The process for the production of a steviol glycoside may be carried outat any suitable pH value. If the recombinant microorganism is yeast, thepH in the fermentation medium preferably has a value of below 6,preferably below 5,5, preferably below 5, preferably below 4,5,preferably below 4, preferably below pH 3,5 or below pH 3,0, or below pH2,5, preferably above pH 2. An advantage of carrying out thefermentation at these low pH values is that growth of contaminantbacteria in the fermentation medium may be prevented.

Such a process may be carried out on an industrial scale.

The product of such a process may be one or more of steviolmonoside,steviolbioside, stevioside or rebaudioside A, rebaudioside B,rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F,rubusoside, dulcoside A. Preferably, rebaudioside A or rebaudioside D isproduced.

Recovery of the diterpene or diterpene glycoside from the resultingbroth may be carried out according to the invention.

In the process for the fermentative production of a steviol glycoside,it may be possible to achieve a concentration of above 5 mg/lfermentation broth, preferably above 10 mg/l, preferably above 20 mg/l,preferably above 30 mg/l fermentation broth, preferably above 40 mg/l,more preferably above 50 mg/l, preferably above 60 mg/l, preferablyabove 70, preferably above 80 mg/l, preferably above 100 mg/l,preferably above 1 g/l, preferably above 5 g/l, preferably above 10 g/l,but usually below 70 g/l in the broth.

As described above, in the event that a diterpene or diterpene glycosideis expressed within the microorganism, such cells may need to be treatedso as to release the steviol glycoside.

The solution and/or composition according to the invention may be usedin any application known for steviol glycosides. In particular, they mayfor instance be used as a sweetener, for example in a food or abeverage. For example steviol glycosides may be formulated in softdrinks, as a tabletop sweetener, chewing gum, dairy product such asyoghurt (eg. plain yoghurt), cake, cereal or cereal-based food,nutraceutical, pharmaceutical, edible gel, confectionery product,cosmetic, toothpastes or other oral cavity composition, etc. Inaddition, a steviol glycoside can be used as a sweetener not only fordrinks, foodstuffs, and other products dedicated for human consumption,but also in animal feed and fodder with improved characteristics.

During the manufacturing of foodstuffs, drinks, pharmaceuticals,cosmetics, table top products, chewing gum the conventional methods suchas mixing, kneading, dissolution, pickling, permeation, percolation,sprinkling, atomizing, infusing and other methods can be used.

The solution and/or composition obtained in this invention can be usedin dry or liquid forms. It can be added before or after heat treatmentof food products. The amount of the sweetener depends on the purpose ofusage. It can be added alone or in the combination with other compounds.

Solutions and compositions produced according to the recovery method ofthe invention may be blended with one or more further non-calorific orcalorific sweeteners. Such blending may be used to improve flavour ortemporal profile or stability. A wide range of both non-calorific andcalorific sweeteners may be suitable for blending with steviolglycosides. For example, non-calorific sweeteners such as mogroside,monatin, aspartame, acesulfame salts, cyclamate, sucralose, saccharinsalts or erythritol. Calorific sweeteners suitable for blending withsteviol glycosides include sugar alcohols and carbohydrates such assucrose, glucose, fructose and HFCS. Sweet tasting amino acids such asglycine, alanine or serine may also be used.

The steviol glycoside can be used in the combination with a sweetenersuppressor, such as a natural sweetener suppressor. It may be combinedwith an umami taste enhancer, such as an amino acid or a salt thereof.

A steviol glycoside can be combined with a polyol or sugar alcohol, acarbohydrate, a physiologically active substance or functionalingredient (for example a carotenoid, dietary fiber, fatty acid,saponin, antioxidant, nutraceutical, flavonoid, isothiocyanate, phenol,plant sterol or stanol (phytosterols and phytostanols), a polyols, aprebiotic, a probiotic, a phytoestrogen, soy protein, sulfides/thiols,amino acids, a protein, a vitamin, a mineral, and/or a substanceclassified based on a health benefits, such as cardiovascular,cholesterol-reducing or anti-inflammatory.

A composition or solution according to the invention may include aflavoring agent, an aroma component, a nucleotide, an organic acid, anorganic acid salt, an inorganic acid, a bitter compound, a protein orprotein hydrolyzate, a surfactant, a flavonoid, an astringent compound,a vitamin, a dietary fiber, an antioxidant, a fatty acid and/or a salt.

A composition or solution of the invention may be applied as a highintensity sweetener to produce zero calorie, reduced calorie or diabeticbeverages and food products with improved taste characteristics. Also itcan be used in drinks, foodstuffs, pharmaceuticals, and other productsin which sugar cannot be used.

In addition, a composition or solution of the invention may be used as asweetener not only for drinks, foodstuffs, and other products dedicatedfor human consumption, but also in animal feed and fodder with improvedcharacteristics.

The examples of products where a composition or solution of theinvention can be used as a sweetening compound can be as alcoholicbeverages such as vodka, wine, beer, liquor, sake, etc; natural juices,refreshing drinks, carbonated soft drinks, diet drinks, zero caloriedrinks, reduced calorie drinks and foods, yogurt drinks, instant juices,instant coffee, powdered types of instant beverages, canned products,syrups, fermented soybean paste, soy sauce, vinegar, dressings,mayonnaise, ketchups, curry, soup, instant bouillon, powdered soy sauce,powdered vinegar, types of biscuits, rice biscuit, crackers, bread,chocolates, caramel, candy, chewing gum, jelly, pudding, preservedfruits and vegetables, fresh cream, jam, marmalade, flower paste,powdered milk, ice cream, sorbet, vegetables and fruits packed inbottles, canned and boiled beans, meat and foods boiled in sweetenedsauce, agricultural vegetable food products, seafood, ham, sausage, fishham, fish sausage, fish paste, deep fried fish products, dried seafoodproducts, frozen food products, preserved seaweed, preserved meat,tobacco, medicinal products, and many others. In principal it can haveunlimited applications.

The sweetened composition comprises a beverage, non-limiting examples ofwhich include non-carbonated and carbonated beverages such as colas,ginger ales, root beers, ciders, fruit-flavored soft drinks (e.g.,citrus-flavored soft drinks such as lemon-lime or orange), powdered softdrinks, and the like; fruit juices originating in fruits or vegetables,fruit juices including squeezed juices or the like, fruit juicescontaining fruit particles, fruit beverages, fruit juice beverages,beverages containing fruit juices, beverages with fruit flavorings,vegetable juices, juices containing vegetables, and mixed juicescontaining fruits and vegetables; sport drinks, energy drinks, nearwater and the like drinks (e.g., water with natural or syntheticflavorants); tea type or favorite type beverages such as coffee, cocoa,black tea, green tea, oolong tea and the like; beverages containing milkcomponents such as milk beverages, coffee containing milk components,cafe au lait, milk tea, fruit milk beverages, drinkable yogurt, lacticacid bacteria beverages or the like; and dairy products.

Generally, the amount of sweetener present in a sweetened compositionvaries widely depending on the particular type of sweetened compositionand its desired sweetness. Those of ordinary skill in the art canreadily discern the appropriate amount of sweetener to put in thesweetened composition.

The composition or solution of the invention obtained as describedherein can be used in dry or liquid forms. It can be added before orafter heat treatment of food products. The amount of the sweetenerdepends on the purpose of usage. It can be added alone or in thecombination with other compounds.

During the manufacturing of foodstuffs, drinks, pharmaceuticals,cosmetics, table top products, chewing gum the conventional methods suchas mixing, kneading, dissolution, pickling, permeation, percolation,sprinkling, atomizing, infusing and other methods can be used.

In solid form, a composition of the present invention can be provided toconsumers in any form suitable for delivery into the comestible to besweetened, including sachets, packets, bulk bags or boxes, cubes,tablets, mists, or dissolvable strips. The composition can be deliveredas a unit dose or in bulk form.

For liquid sweetener systems and compositions convenient ranges offluid, semi-fluid, paste and cream forms, appropriate packing usingappropriate packing material in any shape or form shall be inventedwhich is convenient to carry or dispense or store or transport anycombination containing any of the above sweetener products orcombination of product produced above.

The composition or solution of the invention may include various bulkingagents, functional ingredients, colorants, flavors.

A reference herein to a patent document or other matter which is givenas prior art is not to be taken as an admission that that document ormatter was known or that the information it contains was part of thecommon general knowledge as at the priority date of any of the claims.

The disclosure of each reference set forth herein is incorporated hereinby reference in its entirety.

The present invention is further illustrated by the following Examples:

EXAMPLES General

Standard genetic techniques, such as overexpression of enzymes in thehost cells, as well as for additional genetic modification of hostcells, are known methods in the art, such as described in Sambrook andRussel (2001) “Molecular Cloning: A Laboratory Manual (3^(rd) edition),Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, orF. Ausubel et al, eds., “Current protocols in molecular biology”, GreenPublishing and Wiley Interscience, New York (1987). Methods fortransformation and genetic modification of fungal host cells are knownfrom e.g. EP-A-0 635 574, WO 98/46772, WO 99/60102 and WO 00/37671.

A description of the sequences is set out in Table 1. Sequencesdescribed herein may be defined with reference to the sequence listingor with reference to the database accession numbers also set out inTable 1.

Example 1. Over-Expression of ERG20, BTS1 and tHMG in S. cerevisiae

For over-expression of ERG20, BTS1 tHMG1, expression cassettes weredesigned to be integrated in one locus using technology described inco-pending patent application no. PCT/EP2013/056623. To amplify the 5′and 3′ integration flanks for the integration locus, suitable primersand genomic DNA from a CEN.PK yeast strain (van Dijken et al. Enzyme andMicrobial Technology 26 (2000) 706-714) was used. The different geneswere ordered as cassettes (containing homologous sequence, promoter,gene, terminator, homologous sequence) at DNA2.0. The genes in thesecassettes were flanked by constitutive promoters and terminators. SeeTable 2. Plasmid DNA from DNA2.0 containing the ERG20, tHMG1 and BTS1cassettes were dissolved to a concentration of 100 ng/μl. In a 50 μl PCRmix 20 ng template was used together with 20 pmol of the primers. Thematerial was dissolved to a concentration of 0.5 μg/μl.

TABLE 2 Composition of the over-expression constructs. Promoter ORFTerminator Eno2 Erg20 Adh1 (SEQ ID NO: 201) (SEQ ID NO: 81) (SEQ ID NO:212) Fba1 tHMG1 Adh2 (SEQ ID NO: 202) (SEQ ID NO: 79) (SEQ ID NO: 213)Tef1 Bts1 Gmp1 (SEQ ID NO: 203) (SEQ ID NO: 83) (SEQ ID NO: 214)

For amplification of the selection marker, the pUG7-EcoRV construct(FIG. 1) and suitable primers were used. The KanMX fragment was purifiedfrom gel using the Zymoclean Gel DNA Recovery kit (ZymoResearch). Yeaststrain Cen.PK113-3C was transformed with the fragments listed in Table3.

TABLE 3 DNA fragments used for transformation of ERG20, tHMG1 and BTS1Fragment 5′YPRcTau3 ERG20 cassette tHMG1 cassette KanMX cassatteBTS1 cassette 3′YPRcTau3

After transformation and recovery for 2.5 hours in YEPhD (yeast extractphytone peptone glucose; BBL Phytone Peptone from BD) at 30° C. thecells were plated on YEPhD agar with 200 μg/ml G418 (Sigma). The plateswere incubated at 30° C. for 4 days. Correct integration was establishedwith diagnostic PCR and sequencing. Over-expression was confirmed withLC/MS on the proteins. The schematic of the assembly of ERG20, tHMG1 andBTS1 is illustrated in FIG. 2. This strain is named STV002.

Expression of the CRE-recombinase in this strain led toout-recombination of the KanMX marker. Correct out-recombination, andpresence of ERG20, tHMG and BTS1 was established with diagnostic PCR.

Example 2. Knock Down of Erg9

For reducing the expression of Erg9, an Erg9 knock down construct wasdesigned and used that contains a modified 3′ end, that continues intothe TRP1 promoter driving TRP1 expression.

The construct containing the Erg9-KD fragment was transformed to E. coliTOP10 cells. Transformants were grown in 2PY (2 times Phytone peptoneYeast extract), sAMP medium. Plasmid DNA was isolated with the QIAprepSpin Miniprep kit (Qiagen) and digested with Sall-HF (New EnglandBiolabs). To concentrate, the DNA was precipitated with ethanol. Thefragment was transformed to S. cerevisiae, and colonies were plated onmineral medium (Verduyn et al, 1992. Yeast 8:501-517) agar plateswithout tryptophan. Correct integration of the Erg9-KD construct wasconfirmed with diagnostic PCR and sequencing. The schematic of performedtransformation of the Erg9-KD construct is illustrated in FIG. 3. Thestrain was named STV003.

Example 3. Over-Expression of UGT2 1a

For over-expression of UGT2_1a, technology was used as described inco-pending patent application nos. PCT/EP2013/056623 andPCT/EP2013/055047. The UGT2_1a was ordered as a cassette (containinghomologous sequence, promoter, gene, terminator, homologous sequence) atDNA2.0. For details, see Table 4. To obtain the fragments containing themarker and Cre-recombinase, technology was used as described inco-pending patent application no. PCT/EP2013/055047. The NAT marker,conferring resistance to nourseothricin was used for selection.

TABLE 4 Composition of the over-expression construct Promoter ORFTerminator Pgk1 UGT2_1a Adh2 (SEQ ID NO: 204) (SEQ ID NO: 87) (SEQ IDNO: 213)

Suitable primers were used for amplification. To amplify the 5′ and 3′integration flanks for the integration locus, suitable primers andgenomic DNA from a CEN.PK yeast strain was used.

S. cerevisiae yeast strain STV003 was transformed with the fragmentslisted in Table 5, and the transformation mix was plated on YEPhD agarplates containing 50 μg/ml nourseothricin (Lexy NTC from JenaBioscience).

TABLE 5 DNA fragments used for transformation of UGT2_1a Fragment5′Chr09.01 UGT2_1a cassette NAT-CR RE 3′Chr09.01

Expression of the CRE recombinase is activated by the presence ofgalactose. To induce the expression of the CRE recombinase,transformants were restreaked on YEPh Galactose medium. This resulted inout-recombination of the marker(s) located between lox sites. Correctintegration of the UGT2a and out-recombination of the NAT marker wasconfirmed with diagnostic PCR. The resulting strain was named STV004.The schematic of the performed transformation of the UGT2_1a constructis illustrated in FIG. 4.

Example 4. Over-Expression of Production Pathway to RebA: CPS, KS, KO,KAH, CPR, UGT1, UGT3 and UGT4

All pathway genes leading to the production of RebA were designed to beintegrated in one locus using technology described in co-pending patentapplication no. PCT/EP2013/056623. To amplify the 5′ and 3′ integrationflanks for the integration locus, suitable primers and genomic DNA froma CEN.PK yeast strain was used. The different genes were ordered ascassettes (containing homologous sequence, promoter, gene, terminator,homologous sequence) at DNA2.0 (see Table 5 for overview). The DNA fromDNA2.0 was dissolved to 100 ng/μl. This stock solution was furtherdiluted to 5 ng/μl, of which 1 μl was used in a 50 μl-PCR mixture. Thereaction contained 25 pmol of each primer. After amplification, DNA waspurified with the NucleoSpin 96 PCR Clean-up kit (Macherey-Nagel) oralternatively concentrated using ethanol precipitation.

TABLE 6 Sequences used for production pathway to RebA SEQ Promoter ORFID Terminator Kl prom 12.pro trCPS_SR 61 Sc ADH2.ter (SEQ ID NO: 205)(SEQ ID NO:) Sc PGK1.pro trKS_SR 65 Sc TAL1.ter (SEQ ID NO: 204) (SEQ IDNO: 215) Sc ENO2.pro KO_2 23 Sc TPI1.ter (SEQ ID NO: 201) (SEQ ID NO:216) Ag lox_TEF1.pro KANMX 211 Ag TEF1_lox.ter (SEQ ID NO: 206) (SEQ IDNO: 217) Sc TEF1.pro KAH_4 33 Sc GPM1.ter (SEQ ID NO: 203) (SEQ ID NO:214) Kl prom 6.pro CPR_SR 59 Sc PDC1.ter (SEQ ID NO: 207) (SEQ ID NO:218) Kl prom 3.pro UGT1_SR 71 Sc TDH1.ter (SEQ ID NO: 221) (SEQ ID NO:219) Kl prom 2.pro UGT3_SR 73 Sc ADH1.ter (SEQ ID NO: 222) (SEQ ID NO:212) Sc FBA1.pro UGT4_SR 75 Sc ENO1.ter (SEQ ID NO: 202) (SEQ ID NO:220)

All fragments for the pathway to RebA, the marker and the flanks (seeoverview in Table 7) were transformed to S. cerevisiae yeast strainSTV004. After overnight recovery in YEPhD at 20° C. the transformationmixes were plated on YEPhD agar containing 200 μg/ml G418. These wereincubated 3 days at 25° C. and one night at RT.

TABLE 7 DNA fragments used for transformationof CPS, KS, KO, KanMX, KAH, CPR, UGT1, UGT3 and UGT4. Fragment 5′INT1CPS cassette KS cassette KO cassette KanMX cassette KAH cassetteCPR cassette UGT1 cassette UGT3 cassette UGT4 cassette 3′INT1

Correct integration was confirmed with diagnostic PCR and sequenceanalysis (3500 Genetic Analyzer, Applied Biosystems). The sequencereactions were done with the BigDye Terminator v3.1 Cycle Sequencing kit(Life Technologies). Each reaction (10 μl) contained 50 ng template and3.2 pmol primer. The products were purified by ethanol/EDTAprecipitation, dissolved in 10 μl HiDi formamide and applied onto theapparatus. The strain was named STV016. The schematic of how the pathwayfrom GGPP to RebA is integrated into the genome is illustrated in FIG.5.

Example 5: Construction of Strain STV027

To remove the KanMX marker from the chromosome of strain STV016, thisstrain was transformed with plasmid pSH65, expressing Cre-recombinase(Güldender, 2002). Subsequently plasmid pSH65 was cured from the strainby growing on non-selective medium (YEP 2% glucose). The resulting,KanMX-free and pSH65-free strains, as determined by plating on platescontaining 200 μg G418/ml or 20 μg phleomycin/ml, where no growth shouldoccur, was named STV027. Absence of the KanMX marker was furthermoreconfirmed with diagnostic PCR.

Example 6: Fermentation of Strain STV027

The yeast strain STV027 constructed as described above, was cultivatedin shake-flask (500 ml with 50 ml medium) for 2 days at 30° C. and 280rpm. The medium was based on Verduyn et al. (Verduyn C, Postma E,Scheffers W A, Van Dijken J P. Yeast, 1992 July; 8(7):501-517), withmodifications in the carbon and nitrogen sources, as described in Table8.

TABLE 8 Preculture medium composition Concentration Raw material Formula(g/kg) Galactose C₆H₁₂O₆ 20.0 Urea (NH₂)₂CO 2.3 Potassium dihydrogenphosphate KH₂PO₄ 3.0 Magnesium sulphate MgSO₄•7H₂O 0.5 Trace elementsolution 1 Vitamin solution 1 Concentration Component Formula (g/kg)^(a)Trace elements solution EDTA C₁₀H₁₄N₂Na₂O₈•2H₂O 15.00Zincsulphate•7H₂O ZnSO₄•7H₂O 4.50 Manganesechloride•2H₂O MnCl₂•2H₂O 0.84Cobalt (II) chloride•6H₂O CoCl₂•6H₂O 0.30 Cupper (II) sulphate•5H₂OCuSO₄•5H₂O 0.30 Sodium molybdenum•2H₂O Na₂MoO₄•2H₂O 0.40Calciumchloride•2H₂O CaCl₂•2H₂O 4.50 Ironsulphate•7H₂O FeSO₄•7H₂O 3.00Boric acid H₃BO₃ 1.00 Potassium iodide KI 0.10 ^(b)Vitamin solutionBiotin (D−) C₁₀H₁₆N₂O₃S 0.05 Ca D(+) panthothenate C₁₈H₃₂CaN₂O₁₀ 1.00Nicotinic acid C₆H₅NO₂ 1.00 Myo-inositol C₆H₁₂O₆ 25.00 Thiamine chlorideC₁₂H₁₈Cl₂N₄OS•xH₂O 1.00 hydrochloride Pyridoxol hydrochloride C₈H₁₂ClNO₃1.00 p-aminobenzoic acid C₇H₇NO₂ 0.20

Subsequently, 6 ml of the content of the shake-flask was transferredinto a fermenter (starting volume 0.3 L), which contained the medium asset out in Table 9.

TABLE 9 Composition fermentation medium Final Concentration Raw material(g/kg) Ammonium sulphate (NH₄)₂SO₄ 1 Potassium dihydrogen phosphateKH₂PO₄ 10 Magnesium sulphate MgSO₄•7H₂O 5 Trace element solution — 8Vitamin solution — 8

The pH was controlled at 5.0 by addition of ammonia (12.5 wt %).Temperature was controlled at 27° C. pO₂ was controlled at 40% byadjusting the stirrer speed. Glucose concentration was kept limited bycontrolled feed to the fermenter.

TABLE 10 Composition of the fermentation feed medium Final ConcentrationRaw material Formula (g/kg) Glucose•1aq C₆H₁₂O₆•1aq 330 Potassiumdihydrogen phosphate KH₂PO₄ 10 Magnesium sulphate MgSO₄•7H₂O 5heptahydrate Verduyn trace elements 8 solution Verduyn vitamin solution8

Example 7: Chromatography

Fermentation broth of S. cerevisiae strain STV027 was heat shocked (1 hat 90° C.) and spray dried. Reb A was extracted with ethanol (1stextraction: 1 kg powder with 8 L 90% EtOH, 65° C., contact time 3 h;after filtration, the cake was extracted again with 8 L of 90% EtOH at65° C., contact time 2 h, 1st and 2^(nd) extract were combined). Thisextract was subjected to a 2-step chromatography process to remove othercomponents. In Table 11 the run parameters are displayed.

TABLE 11 chromatography parameters Run 1 Run 2 System: Akta ExplorerAkta Explorer Column: Tricorn 10/20 Tricorn 10/20 Bedvolume and matrix:13.8 ml Diaion 13.8 ml Diaion HP20 HP20 Flow: 150 cm/h 150 cm/h BufferA: Milli Q water Milli Q water Buffer B: 96% Ethanol 96% Ethanol Feed:Extract Reb A in Extract Reb A in 20% EtOH 20% EtOH pH Feed as such:~4.1 pH 8.5 Conductivity n.a. n.a. Load: 500 mg Reb A Elution fraction 2Wash: 20 CV of 20% B 20 CV of 20% B Elution 20-100% B in 18.2 CV 20-100%B in 18.2 CV

The column was loaded with amount of extract corresponding to 500 mg RebA in a 20% EtOH solution, pH kept as such. The column was washed with 20column volumes (CV) of 20% EtOH to wash out unbound components.Subsequently an ethanol gradient from 20% to 100% Buffer B in 18.2 CVwas applied to elute the Reb A. The elution pattern is shown in FIG. 6.Table 12 sets out relative amounts (expressed in %) in differentfractions of the chromatographic run: wash, elution and fractions 1 to6. The initial concentration of the respective compounds is taken as100%.

TABLE 12 Step yields experiment Stepyield Reb-D Reb-A Reb-B Feed 100% 100%  100%  Wash 1 9% 5% 1% Elution 1 3% 2% 0% Elution 2 5% 2% 0%Elution 3 5% 2% 0% Elution 4 4% 2% 1% Elution 5 + 6 32%  73%  63%  Wash2 0% 0% 0% CIP 0.00%   0.02%   0.12%   58%  85%  65% 

After the first purification, the elution fractions were combined anddiluted to 20% ethanol concentration. The pH of this solution is thenadjusted to 8.5 with use of 0.1M NaOH. This solution is used as feed.The elution pattern is shown in FIG. 7. Table 13 then sets out relativeamounts (expressed in %) in different fractions of the chromatographicrun: wash, elution and fractions 1 to 6. The initial concentration ofthe respective compounds is taken as 100%.

TABLE 13 Step yields experiment Stepyield Reb-D Reb-A Reb-B Feed 100% 100%  100%  Flow-through 7% 2% 32%  Wash 1 2% 1% 7% Elution 1 0% 0% 2%Elution 2 0% 0% 1% Elution 3 0% 0% 1% Elution 4 0% 0% 1% Elution 5 130% 77%  48%  Elution 6 10%  26%  6% Wash 2 0% 0% 0% CIP 0% 0% 0% 149% 105%  99% 

Table 14 shows the purity of RebA as % in total dry material. Thestarting material contained 2.3% and the final chromatography fractionsend up at about 30%. That is to say, a 15-fold purification of rebA.

TABLE 14 purification of RebA % Reb A on Fraction Total Dry weight Feed:5x diluted extract 13101 2.30% Fraction 5 + 6 after chromatography pH assuch 20.30%

1. A process for the recovery of one or more steviol glycosides from a steviol glycoside-containing fermentation broth, which method comprises (a) providing a fermentation broth comprising one or more steviol glycosides and one or more non-steviol glycoside components; (b) separating a liquid phase of the broth from a solid phase of the broth; (c) providing an adsorbent resin; (d) contacting the liquid phase of the broth with the adsorbent resin in order to separate at least a portion of the one or more steviol glycosides from the non-steviol glycoside components, thereby to recover one or more steviol glycosides from the fermentation broth containing one or more steviol glycosides.
 2. A process according to claim 1, wherein the adsorbent resin is provided in a packed column.
 3. A process for the recovery of one or more steviol glycosides from a steviol glycoside-containing fermentation broth, which method comprises (a) providing a steviol glycoside-containing fermentation broth; (b) providing an adsorbent resin; (c) contacting the broth with the adsorbent resin in order to separate at least a portion of the one or more steviol glycosides from the non-steviol glycoside components, thereby to recover one or more steviol glycosides from the fermentation broth containing one or more steviol glycosides.
 4. A process according to claim 3, wherein the adsorbent resin is provided in a packed column in an expanded bed mode.
 5. A process according to claim 2, wherein the adsorbent resin is a polystyrene-divinylbenzene resin, a polymethacrylate resin, a polyaromatic resin, a functionalized polymethacrylate-divinybenzene resin, a functionalized polystyrene-divinylbenzene resin or an amino (NH2) bonded methacrylate/divinylbenzene copolymer resin.
 6. A process according to claim 1, wherein the method of separating comprises adsorb/desorb chromatography.
 7. A process according to claim 6, wherein the adsorb/desorb chromatography comprises (a) providing a liquid phase or a broth and a solvent; (b) providing an adsorbent resin; (c) providing an elution solvent; (d) contacting the adsorbent resin with the liquid phase or broth and elution solvent so that at least a portion of the non-steviol glycoside components adsorbs onto the adsorbent enriching the glycoside solution in steviol glycosides and resulting in the formation of a purified steviol glycoside composition that is eluted from the adsorbent along with the elution solvent; and (e) optionally, desorbing the non-steviol glycoside components from the adsorbent.
 8. A process according to claim 7, wherein the adsorbent resin is provided in a packed column.
 9. A process according to claim 7, wherein the elution solvent comprises about 20% weight or less alcohol, optionally comprising ethanol; and about 80% weight or greater water.
 10. A process according to claim 7, wherein the elution solvent comprises about 50% weight or less alcohol, optionally comprising ethanol; and about 50% weight or greater water.
 11. A process according to claim 1, wherein the method of separating comprises fractionation chromatography.
 12. A process according to claim 11, wherein the fractionation chromatography comprises: (a) providing a liquid phase or a broth and a solvent; (b) providing an adsorbent; and (c) contacting the adsorbent with the liquid phase or broth so that at least a portion of non-steviol glycoside components adsorb onto the adsorbent and so that at least a portion of steviol glycoside adsorbs onto the adsorbent, wherein steviol glycosides propagate through the adsorbent at a faster rate than non-steviol glycosides; and (d) collecting a steviol glycoside-containing solution from the adsorbent.
 13. A process according to claim 12, wherein the adsorbent is provided in a packed column.
 14. A process according to claim 12 wherein the solvent comprises about 20% weight or greater alcohol, optionally comprising ethanol, and about 80% weight or less water.
 15. A process according to claim 12, wherein the solvent comprises about 25% to about 35% weight alcohol, optionally comprising ethanol, and about 65% to about 75% water.
 16. A process according to claim 12, wherein the solvent comprises water and wherein the adsorbent is a strongly acidic cationic exchange resin.
 17. A process according to claim 1, wherein the adsorbent has a surface area of about 900 m²/gram or greater.
 18. A process according to claim 6, wherein the adsorbent is functionalized with tertiary amines or quaternary amines.
 19. A process according to claim 1, wherein the recovered steviol glycoside-containing solution has a purity that is at least about 10% greater as compared to a purity of the liquid phase or broth.
 20. A process according to claim 1, wherein the purified steviol glycoside-containing solution comprises, on a dry solids basis, at least about 95% weight of Rebaudioside A, Rebaudioside D or Rebaudioside M.
 21. A process according to claim 1, wherein the steviol glycoside-containing solution is spray-dried to provide a powder.
 22. A solution comprising one or more steviol glycosides obtainable by a process according to claim
 1. 23. A solution according to claim 22 which comprises one or more of steviolmonoside, steviolbioside, stevioside or rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rubusoside, dulcoside A or rebaudioside M.
 24. A solution according to claim 22 which comprises, on a dry solids basis, at least about 95% weight of Rebaudioside A, Rebaudioside D or Rebaudioside M.
 25. A composition which comprises at least about, on a dry solids basis, at least about 95% fermentatively-produced Rebaudioside A, Rebaudioside D or Rebaudioside M.
 26. A composition according to claim 25 which is a powder obtainable by a process wherein the steviol glycoside-containing solution is spray-dried to provide a powder. 